Pretargeting methods and compounds

ABSTRACT

Methods, compounds, compositions and kits that relate to pretargeted delivery of diagnostic and therapeutic agents are disclosed. In particular, methods for radiometal labeling of biotin, as well as related compounds, are described. Articles of manufacture useful in pretargeting methods are also discussed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending PCT PatentApplication No. PCT/US93/05406, filed Jun. 7, 1993 and designating theUnited States, and issued in the United States as U.S. Pat. No.5,608,080 which, in turn, is a continuation-in-part of pending U.S.patent application Ser. No. 07/995,381, filed Dec. 23, 1992 abandoned,which is, in turn, a continuation-in-part of pending U.S. patentapplication Ser. No. 07/895,588, filed Jun. 9, 1992 issued as U.S. Pat.No. 5,283,342.

TECHNICAL FIELD

The present invention relates to methods, compounds, compositions andkits useful for delivering to a target site a targeting moiety that isconjugated to one member of a ligand/anti-ligand pair. Afterlocalization and clearance of the targeting moiety conjugate, direct orindirect binding of a diagnostic or therapeutic agent conjugate at thetarget site occurs. Methods for radiometal labeling of biotin or othersmall molecules, as well as the related compounds, are also disclosed.

BACKGROUND OF THE INVENTION

Conventional cancer therapy is plagued by two problems. The generallyattainable targeting ratio (ratio of administered dose localizing totumor versus administered dose circulating in blood or ratio ofadministered dose localizing to tumor versus administered dose migratingto bone marrow) is low. Also, the absolute dose of radiation ortherapeutic agent delivered to the tumor is insufficient in many casesto elicit a significant tumor response. Improvement in targeting ratioor absolute dose to tumor is sought.

SUMMARY OF THE INVENTION

The present invention is directed to diagnostic and therapeuticpretargeting methods, moieties useful therein and methods of makingthose moieties. Such pretargeting methods are characterized by animproved targeting ratio or increased absolute dose to the target cellsites in comparison to conventional cancer therapy.

The present invention provides radiolabeled small molecules useful indiagnostic or therapeutic pretargeting methods. Certain embodiments ofthe present invention include chelate-biotin compounds and conjugatesincorporating a chelate and a chemically-modified biotin compound usefulin diagnostic or therapeutic pretargeting methods. The present inventionfurther provides methods for making such radiolabeled small molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates blood clearance of biotinylated antibody followingintravenous administration of avidin.

FIG. 2 depicts radiorhenium tumor uptake in a three-step pretargetingprotocol, as compared to administration of radiolabeled antibody(conventional means involving antibody that is covalently linked tochelated radiorhenium).

FIG. 3 depicts the tumor uptake profile of NR-LU-10-streptavidinconjugate (LU-10-StrAv) in comparison to a control profile of nativeNR-LU-10 whole antibody.

FIG. 4 depicts the tumor uptake and blood clearance profiles ofNR-LU-10-streptavidin conjugate.

FIG. 5 depicts the rapid clearance from the blood of asialoorosomucoidin comparison with orosomucoid in terms of percent injected dose ofI-125-labeled protein.

FIG. 6 depicts the 5 minute limited biodistribution of asialoorosomucoidin comparison with orosomucoid in terms of percent injected dose ofI-125-labeled protein.

FIG. 7 depicts NR-LU-10-streptavidin conjugate blood clearance uponadministration of three controls (∘,, ▪) and two doses of a clearingagent (, □) at 25 hours post-conjugate administration.

FIG. 8 shows limited biodistribution data for LU-10-StrAv conjugate uponadministration of three controls (Groups 1, 2 and 5) and two doses ofclearing agent (Groups 3 and 4) at two hours post-clearing agentadministration.

FIG. 9 depicts NR-LU-10-streptavidin conjugate serum biotin bindingcapability at 2 hours post-clearing agent administration.

FIG. 10 depicts NR-LU-10-streptavidin conjugate blood clearance overtime upon administration of a control (∘) and three doses of a clearingagent (∇, Δ, □) at 24 hours post-conjugate administration.

FIG. 11A depicts the blood clearance of LU-10-StrAv conjugate uponadministration of a control (PBS) and three doses (50, 20 and 10 μg) ofclearing agent at two hours post-clearing agent administration.

FIG. 11B depicts LU-10-StrAv conjugate serum biotin binding capabilityupon administration of a control (PBS) and three doses (50, 20 and 10μg) of clearing agent at two hours post-clearing agent administration.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to set forthdefinitions of certain terms to be used within the disclosure.

Targeting moiety: A molecule that binds to a defined population ofcells. The targeting moiety may bind a receptor, an oligonucleotide, anenzymatic substrate, an antigenic determinant, or other binding sitepresent on or in the target cell population. Antibody is used throughoutthe specification as a prototypical example of a targeting moiety. Tumoris used as a prototypical example of a target in describing the presentinvention.

Ligand/anti-ligand pair: A complementary/anti-complementary set ofmolecules that demonstrate specific binding, generally of relativelyhigh affinity. Exemplary ligand/anti-ligand pairs include zinc fingerprotein/dsDNA fragment, enzyme/inhibitor, hapten/antibody,lectin/carbohydrate, ligand/receptor, and biotin/avidin. Biotin/avidinis used throughout the specification as a prototypical example of aligand/anti-ligand pair.

Anti-ligand: As defined herein, an "anti-ligand" demonstrates highaffinity, and preferably, multivalent binding of the complementaryligand. Preferably, the anti-ligand is large enough to avoid rapid renalclearance, and contains sufficient multivalency to accomplishcrosslinking and aggregation of targeting moiety-ligand conjugates.Univalent anti-ligands are also contemplated by the present invention.Anti-ligands of the present invention may exhibit or be derivitized toexhibit structural features that direct the uptake thereof, e.g.,galactose residues that direct liver uptake. Avidin and streptavidin areused herein as prototypical anti-ligands.

Avidin: As defined herein, "avidin" includes avidin, streptavidin andderivatives and analogs thereof that are capable of high affinity,multivalent or univalent binding of biotin.

Ligand: As defined herein, a "ligand" is a relatively small, solublemolecule that exhibits rapid serum, blood and/or whole body clearancewhen administered intravenously in an animal or human. Biotin is used asthe prototypical ligand.

Active Agent: A diagnostic or therapeutic agent ("the payload"),including radionuclides, drugs, anti-tumor agents, toxins and the like.Radionuclide therapeutic agents are used as prototypical active agents.

N_(x) S_(y) Chelates: As defined herein, the term "N_(x) S_(y) chelates"includes bifunctional chelators that are capable of (i) coordinatelybinding a metal or radiometal and (ii) covalently attaching to atargeting moiety, ligand or anti-ligand. Particularly preferred N_(x)S_(y) chelates have N₂ S₂ and N₃ S cores. Exemplary N_(x) S_(y) chelatesare described in Fritzberg et al., Proc. Natl. Acad. Sci. USA85:4024-29, 1988; in Weber et al., Bioconj. Chem. 1:431-37, 1990; and inthe references cited therein, for instance.

Pretargeting: As defined herein, pretargeting involves target sitelocalization of a targeting moiety that is conjugated with one member ofa ligand/anti-ligand pair; after a time period sufficient for optimaltarget-to-non-target accumulation of this targeting moiety conjugate,active agent conjugated to the opposite member of the ligand/anti-ligandpair is administered and is bound (directly or indirectly) to thetargeting moiety conjugate at the target site (two-step pretargeting).Three-step and other related methods described herein are alsoencompassed.

Clearing Agent: An agent capable of binding, completing or otherwiseassociating with an administered moiety (e.g., targeting moiety-ligand,targeting moiety-anti-ligand or anti-ligand alone) present in therecipient's circulation, thereby facilitating circulating moietyclearance from the recipient's body, removal from blood circulation, orinactivation thereof in circulation. The clearing agent is preferablycharacterized by physical properties, such as size, charge,configuration or a combination thereof, that limit clearing agent accessto the population of target cells recognized by a targeting moiety usedin the same treatment protocol as the clearing agent.

Conjugate: A conjugate encompasses chemical conjugates (covalently ornon-covalently bound), fusion proteins and the like.

A recognized disadvantage associated with in vivo administration oftargeting moiety-radioisotopic conjugates for imaging or therapy islocalization of the attached radioactive agent at both non-target andtarget sites. Until the administered radiolabeled conjugate clears fromthe circulation, normal organs and tissues are transitorily exposed tothe attached radioactive agent. For instance, radiolabeled wholeantibodies that are administered in vivo exhibit relatively slow bloodclearance; maximum target site localization generally occurs 1-3 dayspost-administration. Generally, the longer the clearance time of theconjugate from the circulation, the greater the radioexposure ofnon-target organs.

These characteristics are particularly problematic with humanradioimmunotherapy. In human clinical trials, the long circulatinghalf-life of radioisotope bound to whole antibody causes relativelylarge doses of radiation to be delivered to the whole body. Inparticular, the bone marrow, which is very radiosensitive, is thedose-limiting organ of non-specific toxicity.

In order to decrease radioisotope exposure of non-target tissue,potential targeting moieties generally have been screened to identifythose that display minimal non-target reactivity, while retaining targetspecificity and reactivity. By reducing non-target exposure (and adversenon-target localization and/or toxicity), increased doses of aradiotherapeutic conjugate may be administered; moreover, decreasednon-target accumulation of a radiodiagnostic conjugate leads to improvedcontrast between background and target.

Therapeutic drugs, administered alone or as targeted conjugates, areaccompanied by similar disadvantages. Again, the goal is administrationof the highest possible concentration of drug (to maximize exposure oftarget tissue), while remaining below the threshold of unacceptablenormal organ toxicity (due to non-target tissue exposure). Unlikeradioisotopes, however, therapeutic drugs need to be taken into a targetcell to exert a cytotoxic effect. In the case of targetingmoiety-therapeutic drug conjugates, it would be advantageous to combinethe relative target specificity of a targeting moiety with a means forenhanced target cell internalization of the targeting moiety-drugconjugate.

In contrast, enhanced target cell internalization is disadvantageous ifone administers diagnostic agent-targeting moiety conjugates.Internalization of diagnostic conjugates results in cellular catabolismand degradation of the conjugate. Upon degradation, small adducts of thediagnostic agent or the diagnostic agent per se may be released from thecell, thus eliminating the ability to detect the conjugate in atarget-specific manner.

One method for reducing non-target tissue exposure to a diagnostic ortherapeutic agent involves "pretargeting" the targeting moiety at atarget site, and then subsequently administering a rapidly clearingdiagnostic or therapeutic agent conjugate that is capable of binding tothe "pretargeted" targeting moiety at the target site. A description ofsome embodiments of the pretargeting technique may be found in U.S. Pat.No. 4,863,713 (Goodwin et al.).

A typical pretargeting approach ("three-step") is schematically depictedbelow. ##STR1## Briefly, this three-step pretargeting protocol featuresadministration of an antibody-ligand conjugate, which is allowed tolocalize at a target site and to dilute in the circulation. Subsequentlyadministered anti-ligand binds to the antibody-ligand conjugate andclears unbound antibody-ligand conjugate from the blood. Preferredanti-ligands are large and contain sufficient multivalency to accomplishcrosslinking and aggregation of circulating antibody-ligand conjugates.The clearing by anti-ligand is probably attributable to anti-ligandcrosslinking and/or aggregation of antibody-ligand conjugates that arecirculating in the blood, which leads to complex/aggregate clearance bythe recipient's RES (reticuloendothelial system). Anti-ligand clearanceof this type is preferably accomplished with a multivalent molecule;however, a univalent molecule of sufficient size to be cleared by theRES on its own could also be employed. Alternatively, receptor-basedclearance mechanisms, e.g., Ashwell receptor or hexose residue, such asgalactose or mannose residue, recognition mechanisms, may be responsiblefor anti-ligand clearance. Such clearance mechanisms are less dependentupon the valency of the anti-ligand with respect to the ligand than theRES complex/aggregate clearance mechanisms. It is preferred that theligand-anti-ligand pair displays relatively high affinity binding.

A diagnostic or therapeutic agent-ligand conjugate that exhibits rapidwhole body clearance is then administered. When the circulation bringsthe active agent-ligand conjugate in proximity to the target cell-boundantibody-ligand-anti-ligand complex, anti-ligand binds the circulatingactive agent-ligand conjugate and produces anantibody-ligand:anti-ligand:ligand-active agent "sandwich" at the targetsite. Because the diagnostic or therapeutic agent is attached to arapidly clearing ligand (rather than antibody, antibody fragment orother slowly clearing targeting moiety), this technique promisesdecreased non-target exposure to the active agent.

Alternate pretargeting methods eliminate the step of parenterallyadministering an anti-ligand clearing agent. These "two-step" proceduresfeature targeting moiety-ligand or targeting moiety-anti-ligandadministration, followed by administration of active agent conjugated tothe opposite member of the ligand-anti-ligand pair. As an optional step"1.5" in the two-step pretargeting methods of the present invention, aclearing agent (preferably other than ligand or anti-ligand alone) isadministered to facilitate the clearance of circulating targetingmoiety-containing conjugate.

In the two-step pretargeting approach, the clearing agent preferablydoes not become bound to the target cell population, either directly orthrough the previously administered and target cell bound targetingmoiety-anti-ligand or targeting moiety-ligand conjugate. An example oftwo-step pretargeting involves the use of biotinylated human transferrinas a clearing agent for avidin-targeting moiety conjugate, wherein thesize of the clearing agent results in liver clearance oftransferrin-biotin-circulating avidin-targeting moiety complexes andsubstantially precludes association with the avidin-targeting moietyconjugates bound at target cell sites. (See, Goodwin, D. A., Antibod.Immunoconj. Radiopharm., 4: 427-34, 1991).

The two-step pretargeting approach overcomes certain disadvantagesassociated with the use of a clearing agent in a three-step pretargetedprotocol. More specifically, data obtained in animal models demonstratethat in vivo anti-ligand binding to a pretargeted targetingmoiety-ligand conjugate (i.e., the cell-bound conjugate) removes thetargeting moiety-ligand conjugate from the target cell. One explanationfor the observed phenomenon is that the multivalent anti-ligandcrosslinks targeting moiety-ligand conjugates on the cell surface,thereby initiating or facilitating internalization of the resultantcomplex. The apparent loss of targeting moiety-ligand from the cellmight result from internal degradation of the conjugate and/or releaseof active agent from the conjugate (either at the cell surface orintracellularly). An alternative explanation for the observed phenomenonis that permeability changes in the target cell's membrane allowincreased passive diffusion of any molecule into the target cell. Also,some loss of targeting moiety-ligand may result from alteration in theaffinity by subsequent binding of another moiety to the targetingmoiety-ligand, e.g., anti-idiotype monoclonal antibody binding causesremoval of tumor bound monoclonal antibody.

The present invention recognizes that this phenomenon (apparent loss ofthe targeting moiety-ligand from the target cell) may be used toadvantage with regard to in vivo delivery of therapeutic agentsgenerally, or to drug delivery in particular. For instance, a targetingmoiety may be covalently linked to both ligand and therapeutic agent andadministered to a recipient. Subsequent administration of anti-ligandcrosslinks targeting moiety-ligand-therapeutic agent tripartiteconjugates bound at the surface, inducing internalization of thetripartite conjugate (and thus the active agent). Alternatively,targeting moiety-ligand may be delivered to the target cell surface,followed by administration of anti-ligand-therapeutic agent.

In one aspect of the present invention, a targeting moiety-anti-ligandconjugate is administered in vivo; upon target localization of thetargeting moiety-anti-ligand conjugate (i.e., and clearance of thisconjugate from the circulation), an active agent-ligand conjugate isparenterally administered. This method enhances retention of thetargeting moiety-anti-ligand:ligand-active agent complex at the targetcell (as compared with targeting moiety-ligand:anti-ligand:ligand-activeagent complexes and targeting moiety-ligand:anti-ligand-active agentcomplexes). Although a variety of ligand/anti-ligand pairs may besuitable for use within the claimed invention, a preferredligand/anti-ligand pair is biotin/avidin.

In a second aspect of the invention, radioiodinated biotin and relatedmethods are disclosed. Previously, radioiodinated biotin derivativeswere of high molecular weight and were difficult to characterize. Theradioiodinated biotin described herein is a low molecular weightcompound that has been easily and well characterized.

In a third aspect of the invention, a targeting moiety-ligand conjugateis administered in vivo; upon target localization of the targetingmoiety-ligand conjugate (i.e., and clearance of this conjugate from thecirculation), a drug-anti-ligand conjugate is parenterally administered.This two-step method not only provides pretargeting of the targetingmoiety conjugate, but also induces internalization of the subsequenttargeting moiety-ligand-anti-ligand-drug complex within the target cell.Alternatively, another embodiment provides a three-step protocol thatproduces a targeting moiety-ligand:anti-ligand:ligand-drug complex atthe surface, wherein the ligand-drug conjugate is administeredsimultaneously or within a short period of time after administration ofanti-ligand (i.e., before the targeting moiety-ligand-anti-ligandcomplex has been removed from the target cell surface).

In a fourth aspect of the invention, methods for radiolabeling biotinwith technetium-99m, rhenium-186 and rhenium-188 are disclosed.Previously, biotin derivatives were radiolabeled with indium-111 for usein pretargeted immunoscintigraphy (for instance, Virzi et al., Nucl.Med. Biol. 18:719-26, 1991; Kalofonos et al., J. Nucl. Med. 31: 1791-96,1990; Paganelli et al., Canc. Res. 51:5960-66, 1991). However, ^(99m) Tcis a particularly preferred radionuclide for immunoscintigraphy due to(i) low cost, (ii) convenient supply and (iii) favorable nuclearproperties. Rhenium-186 displays chelating chemistry very similar to^(99m) Tc, and is considered to be an excellent therapeutic radionuclide(i.e., a 3.7 day half-life and 1.07 MeV maximum particle that is similarto ¹³¹ I). Therefore, the claimed methods for technetium and rheniumradiolabeling of biotin provide numerous advantages.

The "targeting moiety" of the present invention binds to a definedtarget cell population, such as tumor cells. Preferred targetingmoieties useful in this regard include antibody and antibody fragments,peptides, and hormones. Proteins corresponding to known cell surfacereceptors (including low density lipoproteins, transferrin and insulin),fibrinolytic enzymes, anti-HER2, platelet binding proteins such asannexins, and biological response modifiers (including interleukin,interferon, erythropoietin and colony-stimulating factor) are alsopreferred targeting moieties. Also, anti-EGF receptor antibodies, whichinternalize following binding to the receptor and traffic to the nucleusto an extent, are preferred targeting moieties for use in the presentinvention to facilitate delivery of Auger emitters and nucleus bindingdrugs to target cell nuclei. Oligonucleotides, e.g., antisenseoligonucleotides that are complementary to portions of target cellnucleic acids (DNA or RNA), are also useful as targeting moieties in thepractice of the present invention. Oligonucleotides binding to cellsurfaces are also useful. Analogs of the above-listed targeting moietiesthat retain the capacity to bind to a defined target cell population mayalso be used within the claimed invention. In addition, synthetictargeting moieties may be designed.

Functional equivalents of the aforementioned molecules are also usefulas targeting moieties of the present invention. One targeting moietyfunctional equivalent is a "mimetic" compound, an organic chemicalconstruct designed to mimic the proper configuration and/or orientationfor targeting moiety-target cell binding. Another targeting moietyfunctional equivalent is a short polypeptide designated as a "minimal"polypeptide, constructed using computer-assisted molecular modeling andmutants having altered binding affinity, which minimal polypeptidesexhibit the binding affinity of the targeting moiety.

Preferred targeting moieties of the present invention are antibodies(polyclonal or monoclonal), peptides, oligonucleotides or the like.Polyclonal antibodies useful in the practice of the present inventionare polyclonal (Vial and Callahan, Univ. Mich. Med. Bull., 20: 284-6,1956), affinity-purified polyclonal or fragments thereof (Chao et al.,Res. Comm. in Chem. Path. & Pharm., 9: 749-61, 1974).

Monoclonal antibodies useful in the practice of the present inventioninclude whole antibody and fragments thereof. Such monoclonal antibodiesand fragments are producible in accordance with conventional techniques,such as hybridoma synthesis, recombinant DNA techniques and proteinsynthesis. Useful monoclonal antibodies and fragments may be derivedfrom any species (including humans) or may be formed as chimericproteins which employ sequences from more than one species. See,generally, Kohler and Milstein, Nature, 256: 495-97, 1975; Eur. J.Immunol., 6: 511-19, 1976.

Human monoclonal antibodies or "humanized" murine antibody are alsouseful as targeting moieties in accordance with the present invention.For example, murine monoclonal antibody may be "humanized" bygenetically recombining the nucleotide sequence encoding the murine Fvregion (i.e., containing the antigen binding sites) or thecomplementarity determining regions thereof with the nucleotide sequenceencoding a human constant domain region and an Fc region, e.g., in amanner similar to that disclosed in European Patent Application No.0,411,893 A2. Some murine residues may also be retained within the humanvariable region framework domains to ensure proper target site bindingcharacteristics. Humanized targeting moieties are recognized to decreasethe immunoreactivity of the antibody or polypeptide in the hostrecipient, permitting an increase in the half-life and a reduction inthe possibility of adverse immune reactions.

Types of active agents (diagnostic or therapeutic) useful herein includetoxins, anti-tumor agents, drugs and radionuclides. Several of thepotent toxins useful within the present invention consist of an A and aB chain. The A chain is the cytotoxic portion and the B chain is thereceptor-binding portion of the intact toxin molecule (holotoxin).Because toxin B chain may mediate non-target cell binding, it is oftenadvantageous to conjugate only the toxin A chain to a targeting protein.However, while elimination of the toxin B chain decreases non-specificcytotoxicity, it also generally leads to decreased potency of the toxinA chain-targeting protein conjugate, as compared to the correspondingholotoxin-targeting protein conjugate.

Preferred toxins in this regard include holotoxins, such as abrin,ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin, pertussistoxin and Shiga toxin; and A chain or "A chain-like" molecules, such asricin A chain, abrin A chain, modeccin A chain, the enzymatic portion ofPseudomonas exotoxin A, Diphtheria toxin A chain, the enzymatic portionof pertussis toxin, the enzymatic portion of Shiga toxin, gelonin,pokeweed antiviral protein, saporin, tritin, barley toxin and snakevenom peptides. Ribosomal inactivating proteins (RIPs), naturallyoccurring protein synthesis inhibitors that lack translocating andcell-binding ability, are also suitable for use herein. Extremely highlytoxic toxins, such as palytoxin and the like, are also contemplated foruse in the practice of the present invention.

Preferred drugs suitable for use herein include conventionalchemotherapeutics, such as vinblastine, doxorubicin, bleomycin,methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine,cyclophosphamide and cisplatinum, as well as other conventionalchemotherapeutics as described in Cancer: Principles and Practice ofOncology, 2d ed., V. T. DeVita, Jr., S. Hellman, S. A. Rosenberg, J. B.Lippincott Co., Philadelphia, Pa., 1985, Chapter 14. A particularlypreferred drug within the present invention is a trichothecene.

Trichothecenes are drugs produced by soil fungi of the class Fungiimperfecti or isolated from Baccharus megapotamica (Bamburg, J. R. Proc.Molec. Subcell. Biol. 8:41-110, 1983; Jarvis & Mazzola, Acc. Chem. Res.15:338-395, 1982). They appear to be the most toxic molecules thatcontain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Orq.Naturst. 31:61-117, 1974). They are all reported to act at the level ofthe ribosome as inhibitors of protein synthesis at the initiation,elongation, or termination phases.

There are two broad classes of trichothecenes: those that have only acentral sesquiterpenoid structure and those that have an additionalmacrocyclic ring (simple and macrocyclic trichothecenes, respectively).The simple trichothecenes may be subdivided into three groups (i.e.,Group A, B, and C) as described in U.S. Pat. Nos. 4,744,981 and4,906,452 (incorporated herein by reference). Representative examples ofGroup A simple trichothecenes include: Scirpene, Roridin C,dihydrotrichothecene, Scirpen-4, 8-diol, Verrucarol, Scirpentriol, T-2tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol, trichodermin,deacetylcalonectrin, calonectrin, diacetylverrucarol,4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol,7-hydroxydiacetoxyscirpenol, 8-hydroxydiacetoxy-scirpenol (Neosolaniol),7,8-dihydroxydiacetoxyscirpenol, 7-hydroxy-8-acetyldiacetoxyscirpenol,8-acetylneosolaniol, NT-1, NT-2, HT-2, T-2, and acetyl T-2 toxin.Representative examples of Group B simple trichothecenes include:Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol,5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol,4-acetylnivalenol (Fusarenon-X), 4,15-idacetylnivalenol,4,7,15-triacetylnivalenol, and tetra-acetylnivalenol. Representativeexamples of Group C simple trichothecenes include: Crotocol andCrotocin. Representative macrocyclic trichothecenes include VerrucarinA, Verrucarin B, Verrucarin J (Satratoxin C), Roridin A, Roridin D,Roridin E (Satratoxin D), Roridin H, Satratoxin F, Satratoxin G,Satratoxin H, Vertisporin, Mytoxin A, Mytoxin C, Mytoxin B, Myrotoxin A,Myrotoxin B, Myrotoxin C, Myrotoxin D, Roritoxin A, Roritoxin B, andRoritoxin D. In addition, the general "trichothecene" sesquiterpenoidring structure is also present in compounds termed "baccharins" isolatedfrom the higher plant Baccharis megapotamica, and these are described inthe literature, for instance as disclosed by Jarvis et al. (Chemistry ofAlleopathy, ACS Symposium Series No. 268: ed. A. C. Thompson, 1984, pp.149-159).

Experimental drugs, such as mercaptopurine, N-methylformamide,2-amino-1,3,4-thiadiazole, melphalan, hexamethylmelamine, galliumnitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin,bromodeoxyuridine, iododeoxyuridine, semustine,1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea,N,N'-hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwiniaasparaginase, ifosfamide, 2-mercaptoethane sulfonate, teniposide, taxol,3-deazauridine, soluble Baker's antifol, homoharringtonine,cyclocytidine, acivicin, ICRF-187, spiromustine, levamisole,chlorozotocin, aziridinyl benzoquinone, spirogermanium, aclarubicin,pentostatin, PALA, carboplatin, amsacrine, caracemide, iproplatin,misonidazole, dihydro-5-azacytidine, 4'-deoxy-doxorubicin, menogaril,triciribine phosphate, fazarabine, tiazofurin, teroxirone, ethiofos,N-(2-hydroxyethyl)-2-nitro-1H-imidazole-1-acetamide, mitoxantrone,acodazole, amonafide, fludarabine phosphate, pibenzimol, didemnin B,merbarone, dihydrolenperone, flavone-8-acetic acid, oxantrazole,ipomeanol, trimetrexate, deoxyspergualin, echinomycin, anddideoxycytidine (see NCI Investigational Drugs, Pharmaceutical Data1987, NIH Publication No. 88-2141, Revised November 1987) are alsopreferred.

Radionuclides useful within the present invention includegamma-emitters, positron-emitters, Auger electron-emitters, X-rayemitters and fluorescence-emitters, with beta- or alpha-emitterspreferred for therapeutic use. Radionuclides are well-known in the artand include ¹²³ I, ¹²⁵ I, ¹³⁰ I, ¹³¹ I, ¹³³ I, ¹³⁵ I, ⁴⁷ Sc, ⁷² As, ⁷²Se, ⁹⁰ Y, ⁸⁸ Y, ⁹⁷ Ru, ¹⁰⁰ Pd, ^(101m) Rh, ¹¹⁹ Sb, ¹²⁸ Ba, ¹⁹⁷ Hg, ²¹¹At, ²¹² Bi, ¹⁵³ Sm, ¹⁶⁹ Eu, ²¹² Pb, ¹⁰⁹ Pd, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁷ Cu,⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^(99m) Tc, ¹¹ C, ¹³ N, ¹⁵ O and ¹⁸ f. Preferredtherapeutic radionuclides include ¹⁸⁸ Re, ¹⁸⁶ Re, ²⁰³ Pb, ²¹² Pb, ²¹²Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ⁷⁷ Br, ²¹¹ At, ⁹⁷ Ru, ¹⁰⁵Rh, ¹⁹⁸ Au and ¹⁹⁹ Ag or ¹⁷⁷ Lu.

Other anti-tumor agents,e.g., agents active against proliferating cells,are administrable in accordance with the present invention. Exemplaryanti-tumor agents include cytokines, such as IL-2, tumor necrosis factoror the like, lectin inflammatory response promoters (selectins), such asL-selectin, E-selectin, P-selectin or the like, and like molecules.

Ligands suitable for use within the present invention include biotin,haptens, lectins, epitopes, dsDNA fragments, enzyme inhibitors andanalogs and derivatives thereof. Useful complementary anti-ligandsinclude avidin (for biotin), carbohydrates (for lectins) and antibody,fragments or analogs thereof, including mimetics (for haptens andepitopes) and zinc finger proteins (for dsDNA fragments) and enzymes(for enzyme inhibitors). Preferred ligands and anti-ligands bind to eachother with an affinity of at least about k_(D) ≧10⁹ M.

The 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetra acetic acid(DOTA)-biotin conjugate (DOTA-LC-biotin) depicted below has beenreported to have desirable in vivo biodistribution and is clearedprimarily by renal excretion. ##STR2## DOTA may also be conjugated toother ligands or to anti-ligands in the practice of the presentinvention.

Because DOTA strongly binds Y-90 and other radionuclides, it has beenproposed for use in radioimmunotherapy. For therapy, it is veryimportant that the radionuclide be stably bound within the DOTA chelateand that the DOTA chelate be stably attached to a ligand or anti-ligand.For illustrative purposes, DOTA-biotin conjugates are described. Onlyradiolabeled DOTA-biotin conjugates exhibiting those two characteristicsare useful to deliver radionuclides to the targets. Release of theradionuclide from the DOTA chelate or cleavage of the biotin and DOTAconjugate components in serum or at non-target sites renders theconjugate unsuitable for use in therapy.

Serum stability of DOTA-LC-biotin (where LC refers to the "long chain"linker, including an aminocaproyl spacer between the biotin and the DOTAconjugate components) shown above, while reported in the literature tobe good, has proven to be problematic. Experimentation has revealed thatDOTA-LC-biotin is rapidly cleared from the blood and excreted into theurine as fragments, wherein the biotinamide bond rather than theDOTA-amide bond has been cleaved, as shown below. ##STR3##

Additional experimentation employing PIP-biocytin conjugates producedparallel results as shown below. ##STR4## Cleavage of the benzamide wasnot observed as evidenced by the absence of detectable quantities ofiodobenzoic acid in the serum.

It appears that the cleavage results from the action of serumbiotinidase. Biotinidase is a hydrolytic enzyme that catalyzes thecleavage of biotin from biotinyl peptides. See, for example,Evangelatos, et al., "Biotinidase Radioassay Using an I-125-BiotinDerivative, Avidin, and Polyethylene Glycol Reagents," AnalyticalBiochemistry, 196: 385-89, 1991.

Drug-biotin conjugates which structurally resemble biotinyl peptides arepotential substrates for cleavage by plasma biotinidase. Poor in vivostability therefore limits the use of drug-biotin conjugates intherapeutic applications. The use of peptide surrogates to overcome poorstability of peptide therapeutic agents has been an area of intenseresearch effort. See, for example, Spatola, Peptide BackboneModification: A Structure-Activity Analysis of Peptide Containing AmideBond Surrogates, "Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins," vol. 7, Weinstein, ed., Marcel Dekker, New York, 1983;and Kim et al., "A New Peptide Bond Surrogate: 2-Isoxazoline inPseudodipeptide Chemistry," Tetrahedron Letters, 45: 6811-14, 1991.

Elimination of the aminocaproyl spacer of DOTA-LC-biotin givesDOTA-SC-biotin (where the SC indicates the "short chain" linker betweenthe DOTA and biotin conjugate components), which molecule is shownbelow: ##STR5## DOTA-SC-biotin exhibits significantly improved serumstability in comparison to DOTA-LC-biotin. This result does not appearto be explainable on the basis of biotinidase activity alone. Theexperimentation leading to this conclusion is summarized in the Tableset forth below.

    ______________________________________                                        Time Dependent Cleavage of DOTA-Biotin Conjugates                                       % Avidin Binding                                                                         Y-90-LC     Y-90-SC                                        Time at 37° C. PIP-Biocytin DOTA-Biotin DOTA-Biotin                  ______________________________________                                        5 Minutes 75%        50%         --                                             15 Minutes 57% 14% --                                                         30 Minutes 31% 12% --                                                         60 Minutes -- 0% 98%                                                          20 Hours -- 0% 60%                                                          ______________________________________                                         where "--" indicates that the value was not measured.                    

The difference in serum stability between DOTA-LC-biotin andDOTA-SC-biotin might be explained by the fact that the SC derivativecontains an aromatic amide linkage in contrast to the aliphatic amidelinkage of the LC derivative, with the aliphatic amide linkage beingmore readily recognized by enzymes as a substrate therefor. Thisargument cannot apply to biotinidase, however, because biotinidase veryefficiently cleaves aromatic amides. In fact, it is recognized that thesimplest and most commonly employed biotinidase activity measuringmethod uses N-(d-biotinyl)-4-aminobenzoate (BPABA) as a substrate, withthe hydrolysis of BPABA resulting in the liberation of biotin and4-aminobenzoate (PABA). See, for example, B. Wolf, et al., "Methods inEnzymology," pp. 103-111, Academic Press Inc., 1990. Consequently, onewould predict that DOTA-SC-biotin, like its LC counterpart, would be abiotinidase substrate. Since DOTA-SC-biotin exhibits serum stability,biotinidase activity alone does not adequately explain why someconjugates are serum stable while others are not. A series ofDOTA-biotin conjugates was therefore synthesized by the presentinventors to determine which structural features conferred serumstability to the conjugates.

Some general strategies for improving serum stability of peptides withrespect to enzymatic action are the following: incorporation of D-aminoacids, N-methyl amino acids and alpha-substituted amino acids.

In vivo stable biotin-DOTA conjugates are useful within the practice ofthe present invention. In vivo stability imparts the followingadvantages:

1) increased tumor uptake in that more of the radioisotope will betargeted to the previously localized targeting moiety-streptavidin; and

2) increased tumor retention, if biotin is more stably bound to theradioisotope. In addition, the linkage between DOTA and biotin may alsohave a significant impact on biodistribution (including normal organuptake, target uptake and the like) and pharmacokinetics.

The strategy for design of the DOTA-containing molecules and conjugatesof the present invention involved three primary considerations:

1) in vivo stability (including biotinidase and general peptidaseactivity resistance), with an initial acceptance criterion of 100%stability for 1 hour;

2) renal excretion; and

3) ease of synthesis.

The DOTA-biotin conjugates of the present invention reflect theimplementation of one or more of the following strategies:

1) substitution of the carbon adjacent to the cleavage susceptible amidenitrogen;

2) alkylation of the cleavage susceptible amide nitrogen;

3) substitution of the amide carbonyl with an alkyl amino group;

4) incorporation of D-amino acids as well as analogs or derivativesthereof; or

5) incorporation of thiourea linkages.

DOTA-biotin conjugates in accordance with the present invention may begenerally characterized as follows: conjugates that retain the biotincarboxy group in the structure thereof and those that do not (i.e., theterminal carboxy group of biotin has been reduced or otherwisechemically modified. Structures of such conjugates represented by thefollowing general formula have been devised: ##STR6## wherein L mayalternatively be substituted in one of the following ways on one of the--CH₂ --COOH branches of the DOTA structure: --CH(L)--COOH or --CH₂ COOLor --CH₂ COL). When these alternative structures are employed, theportion of the linker bearing the functional group for binding with theDOTA conjugate component is selected for the capability to interact witheither the carbon or the carboxy in the branch portions of the DOTAstructure, with the serum stability conferring portion of the linkerstructure being selected as described below.

In the case where the linkage is formed on the core of the DOTAstructure as shown above, L is selected according to the followingprinciples, with the portion of the linker designed to bind to the DOTAconjugate component selected for the capability to bind to an amine.

A. One embodiment of the present invention includes linkersincorporating a D-amino acid spacer between a DOTA aniline amine and thebiotin carboxy group shown above. Substituted amino acids are preferredfor these embodiments of the present invention, becausealpha-substitution also confers enzymatic cleavage resistance. ExemplaryL moieties of this embodiment of the present invention may berepresented as follows: ##STR7## where R¹ is selected from lower alkyl,lower alkyl substituted with hydrophilic groups (preferably, ##STR8##where n is 1 or 2), glucuronide-substituted amino acids or otherglucuronide derivatives; and

R² is selected from hydrogen, lower alkyl, substituted lower alkyl(e.g., hydroxy, sulfate, phosphonate or a hydrophilic moiety (preferablyOH).

For the purposes of the present disclosure, the term "lower alkyl"indicates an alkyl group with from one to five carbon atoms. Also, theterm "substituted" includes one or several substituent groups, with asingle substituent group preferred.

Preferred L groups of this embodiment of the present invention includethe following:

R¹ =CH₃ and R² =H (a D-alanine derivative, with a synthetic schemetherefor shown in Example XV);

R¹ =CH₃ and R² =CH₃ (an N-methyl-D-alanine derivative);

R¹ =CH₂ --OH and R² =H (a D-serine derivative);

R¹ =CH₂ OSO₃ and R² =H (a D-serine-O-sulfate-derivative); and ##STR9##and R² =H (a D-serine-O-phosphonate-derivative);

Other preferred moieties of this embodiment of the present inventioninclude molecules wherein R¹ is hydrogen and R² =--(CH₂)_(n) OH or asulfate or phosphonate derivative thereof and n is 1 or 2 as well asmolecules wherein R¹ is ##STR10##

Preferred moieties incorporating the glucuronide of D-lysine and theglucuronide of amino pimelate are shown below as I and II, respectively.##STR11##

A particularly preferred linker of this embodiment of the presentinvention is the D-alanine derivative set forth above.

B. Linkers incorporating alkyl substitution on one or more amidenitrogen atoms are also encompassed by the present invention, with someembodiments of such linkers preparable from L-amino acids. Amide bondshaving a substituted amine moiety are less susceptible to enzymaticcleavage. Such linkers exhibit the following general formula: ##STR12##where R⁴ is selected from hydrogen, lower alkyl, lower alkyl substitutedwith hydroxy, sulfate, phosphonate or the like and ##STR13## R₃ isselected from hydrogen; an amine; lower alkyl; an amino- or a hydroxy-,sulfate- or phosphonate-substituted lower alkyl; a glucuronide or aglucuronide-derivatized amino groups; and

n ranges from 0-4.

Preferred linkers of this embodiment of the present invention include:

R³ =H and R⁴ =CH₃ when n=4, synthesizable as discussed in Example XV;

R³ =H and R⁴ =CH₃ when n=0, synthesizable from N-methyl-glycine (havinga trivial name of sarcosine) as described in Example XV;

R³ =NH₂ and R⁴ =CH₃, when n=0;

R³ =H and ##STR14## when n=4 (Bis-DOTA-LC-biotin), synthesizable frombromohexanoic acid as discussed in Example XV; and

R³ =H and ##STR15## when n=0 (bis-DOTA-SC-biotin), synthesizable fromiminodiacetic acid.

The synthesis of a conjugate including a linker wherein R³ is H and R⁴is --CH₂ CH₂ OH and n is 0 is also described in Example XV.Schematically, the synthesis of a conjugate of this embodiment of thepresent invention wherein n is 0, R³ is H and R⁴ is --CH₂ --COOH isshown below. ##STR16##

Bis-DOTA-LC-biotin, for example, offers the following advantages:

1) incorporation of two DOTA molecules on one biotin moiety increasesthe overall hydrophilicity of the biotin conjugate and thereby directsin vivo distribution to urinary excretion; and

2) substitution of the amide nitrogen adjacent to the biotin carboxylgroup blocks peptide and/or biotinidase cleavage at that site.

Bis-DOTA-LC-biotin, the glycine-based linker and the N-methylated linkerwhere R³ =H, R⁴ =CH₃, n=4 are particularly preferred linkers of thisembodiment of the present invention.

C. Another linker embodiment incorporates a thiourea moiety therein.Exemplary thiourea adducts of the present invention exhibit thefollowing general formula: ##STR17## where R⁵ is selected from hydrogenor lower alkyl; R⁶ is selected from H and a hydrophilic moiety; and

n ranges from 0-4.

Preferred linkers of this embodiment of the present invention are asfollows:

R⁵ =H and R⁶ =H when n=5;

R⁵ =H and R⁶ =COOH when n=5; and

R⁵ =CH₃ and R⁶ =COOH when n=5.

The second preferred linker recited above can be prepared using eitherL-lysine or D-lysine.

Similarly, the third preferred linker can be prepared using eitherN-methyl-D-lysine or N-methyl-L-lysine.

Another thiourea adduct of minimized lipophilicity is ##STR18## whichmay be formed via the addition of biotinhydrazide (commerciallyavailable from Sigma Chemical Co., St. Louis, Mo.) andDOTA-benzylisothiocyanate (a known compound synthesized in one step fromDOTA-aniline), with the thiourea-containing compound formed as shownbelow. ##STR19##

D. Amino acid-derived linkers of the present invention with substitutionof the carbon adjacent to the cleavage susceptible amide have thegeneral formula set forth below: ##STR20## wherein Z is --(CH₂)₂ --,conveniently synthesized form glutamic acid; or

Z=--CH₂ --S--CH₂ --, synthesizable from cysteine and iodo-acetic acid;or

Z=--CH₂ --, conveniently synthesized form aspartic acid; or

Z=--(CH₂)_(n) --CO--O--CH₂ --, where n ranges from 1-4 and which issynthesizable from serine.

E. Another exemplary linker embodiment of the present invention has thegeneral formula set forth below: ##STR21## and n ranges from 1-5.

F. Another embodiment involves disulfidecontaining linkers, whichprovide a metabolically cleavable moiety (--S--S--) to reduce non-targetretention of the biotin-DOTA conjugate. Exemplary linkers of this typeexhibit the following formula: ##STR22## where n and n' preferably rangebetween 0 and 5.

The advantage of using conditionally cleavable linkers is an improvementin target/non-target localization of the active agent. Conditionallycleavable linkers include enzymatically cleavable linkers, linkers thatare cleaved under acidic conditions, linkers that are cleaved underbasic conditions and the like. More specifically, use of linkers thatare cleaved by enzymes, which are present in non-target tissues butreduced in amount or absent in target tissue, can increase target cellretention of active agent relative to non-target cell retention. Suchconditionally cleavable linkers are useful, for example, in deliveringtherapeutic radionuclides to target cells, because such active agents donot require internalization for efficacy, provided that the linker isstable at the target cell surface or protected from target celldegradation.

Cleavable linkers are also useful to effect target site selectiverelease of active agent at target sites. Active agents that arepreferred for cleavable linker embodiments of the present invention arethose that are substantially non-cytotoxic when conjugated to ligand oranti-ligand. Such active agents therefore require release from theligand- or anti-ligand-containing conjugate to gain full potency. Forexample, such active agents, while conjugated, may be unable to bind toa cell surface receptor; unable to internalize either actively orpassively; or unable to serve as a binding substrate for a soluble(intra- or inter-cellular) binding protein or enzyme. Exemplary of anactive agent-containing conjugate of this type is chemotherapeuticdrug-cis-aconityl-biotin. The cis-aconityl linker is acid sensitive.Other acid sensitive linkers useful in cleavable linker embodiments ofthe present invention include esters, thioesters and the like. Use ofconjugates wherein an active agent and a ligand or an anti-ligand arejoined by a cleavable linker will result in the selective release of theactive agent at tumor cell target sites, for example, because theinter-cellular milieu of tumor tissue is generally of a lower pH (morehighly acidic) than the inter-cellular milieu of normal tissue.

G. Ether, thioether, ester and thioester linkers are also useful in thepractice of the present invention. Ether and thioether linkers arestable to acid and basic conditions and are therefore useful to deliveractive agents that are potent in conjugated form, such as radionuclidesand the like. Ester and thioesters are hydrolytically cleaved underacidic or basic conditions or are cleavable by enzymes includingesterases, and therefore facilitate improved target:non-targetretention. Exemplary linkers of this type have the following generalformula: ##STR23## where X is O or S; and

Q is a bond, a methylene group, a --CO-- group or --CO--(CH₂)_(n)--NH--; and

n ranges from 1-5.

Other such linkers have the general formula:

--CH₂ --X--Q, where Q and X are defined as set forth above.

H. Another amino-containing linker of the present invention isstructured as follows: ##STR24## where R⁷ is lower alkyl, preferablymethyl. In this case, resistance to enzymatic cleavage is conferred bythe alkyl substitution on the amine.

I. Polymeric linkers are also contemplated by the present invention.Dextran and cyclodextran are preferred polymers useful in thisembodiment of the present invention as a result of the hydrophilicity ofthe polymer, which leads to favorable excretion of conjugates containingthe same. Other advantages of using dextran polymers are that suchpolymers are substantially non-toxic and non-immunogenic, that they arecommercially available in a variety of sizes and that they are easy toconjugate to other relevant molecules. Also, dextran-linked conjugatesexhibit advantages when non-target sites are accessible to dextranase,an enzyme capable of cleaving dextran polymers into smaller units whilenon-target sites are not so accessible.

Other linkers of the present invention are produced prior to conjugationto DOTA and following the reduction of the biotin carboxy moiety. Theselinkers of the present invention have the following general formula:##STR25##

Embodiments of linkers of this aspect of the present invention includethe following:

J. An ether linkage as shown below may be formed in a DOTA-biotinconjugate in accordance with the procedure indicated below.

    L'=--NH--CO--(CH.sub.2).sub.n --O--

where n ranges from 1 to 5, with 1 preferred. ##STR26## This linker hasonly one amide moiety which is bound directly to the DOTA aniline (as inthe structure of DOTA-SC-biotin). In addition, the ether linkage impartshydrophilicity, an important factor in facilitating renal excretion.

K. An amine linker formed from reduced biotin (hydroxybiotin oraminobiotin) is shown below, with conjugates containing such a linkerformed, for example, in accordance with the procedure described inExample XV.

    L'=--NH--

This linker contains no amide moieties and the unalkylated amine mayimpart favorable biodistribution properties since unalkylatedDOTA-aniline displays excellent renal clearance.

L. Substituted amine linkers, which can form conjugates via amino-biotinintermediates, are shown below. ##STR27## where R⁸ is H; --(CH₂)₂ --OHor a sulfate or phosphonate derivative thereof; or ##STR28## or thelike; and R⁹ is a bond or --(CH₂)_(n) --CO--NH--, where n ranges from0-5 and is preferably 1 and where q is 0 or 1. These moieties exhibitthe advantages of an amide only directly attached to DOTA-aniline andeither a nonamide amine imparting a positive charge to the linker invivo or a N-alkylated glucuronide hydrophilic group, each alternativefavoring renal excretion.

M. Amino biotin may also be used as an intermediate in the production ofconjugates linked by linkers having favorable properties, such as athiourea-containing linker of the formula:

    L'=--NH--CS--NH--

Conjugates containing this thiourea linker have the followingadvantages: no cleavable amide and a short, fairly polar linker whichfavors renal excretion.

A bis-DOTA derivative of the following formula can also be formed fromamino-biotin. ##STR29## where n ranges from 1 to 5, with 1 and 5preferred. This molecule offers the advantages of the previouslydiscussed bis-DOTA derivatives with the added advantage of no cleavableamides.

Additional linkers of the present invention which are employed in theproduction of conjugates characterized by a reduced biotin carboxymoiety are the following:

L=--(CH₂)₄ --NH--, wherein the amine group is attached to the methylenegroup corresponding to the reduced biotin carboxy moiety and themethylene chain is attached to a core carbon in the DOTA ring. Such alinker is conveniently synthesizable from lysine.

L=--(CH₂)_(q) --CO--NH--, wherein q is 1 or 2, and wherein the aminegroup is attached to the methylene group corresponding to the reducedbiotin carboxy moiety and the methylene group(s) are attached to a corecarbon in the DOTA ring. This moiety is synthesizable from amino-biotin.

The linkers set forth above are useful to produce conjugates having oneor more of the following advantages:

bind avidin or streptavidin with the same or substantially similaraffinity as free biotin;

bind metal M⁺³ ions efficiently and with high kinetic stability;

are excreted primarily through the kidneys into urine;

are stable to bodily fluid amidases;

penetrate tissue rapidly and bind to pretargeted avidin or streptavidin;and

are excreted rapidly with a whole body residence half-life of less thanabout 5 hours.

Synthetic routes to an intermediate of the DOTA-biotin conjugatesdepicted above, nitrobenzyl-DOTA, have been proposed. These proposedsynthetic routes produce the intermediate compound in suboptimal yield,however. For example, Renn and Meares, "Large Scale Synthesis ofBifunctional Chelating AgentQ-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid, and the Determination of its Enantiomeric Purity by ChiralChromatography," Bioconj. Chem., 3: 563-9, 1992, describe a nine-stepsynthesis of nitrobenzyl-DOTA, including reaction steps that eitherproceed in low yield or involve cumbersome transformations orpurifications. More specifically, the sixth step proceeds in only 26%yield, and the product must be purified by preparative HPLC.Additionally, step eight proceeds in good yield, but the processinvolves copious volumes of the coreactants.

These difficulties in steps 6-8 of the prior art synthesis are overcomein the practice of the present invention through the use of thefollowing synthetic alternative therefor. ##STR30##

The poor yield in step six of the prior art synthesis procedure, inwhich a tetra amine alcohol is converted to a tetra-toluenesulfonamidetoluenesulfonate as shown below, is the likely result of prematureformation of the O-toluenesulfonate functionality (before all of theamine groups have been converted to their corresponding sulfonamides.##STR31## Such a sequence of events would potentially result in unwantedintra- or inter-molecular displacement of the reactiveO-toluenesulfonate by unprotected amine groups, thereby generatingnumerous undesirable side-products.

This problem is overcome in the aforementioned alternative synthesisscheme of the present invention by reacting the tetra-amine alcohol withtrifluoroacetic anhydride. Trifluoroacetates, being much poorer leavinggroups than toluenesulfonates, are not vulnerable to analogous sidereactions. In fact, the easy hydrolysis of trifluoroacetate groups, asreported in Greene and Wuts, "Protecting Groups in Organic Synthesis,"John Wiley and Sons, Inc., New York, p. 94, 1991, suggests that additionof methanol to the reaction mixture following consumption of all aminesshould afford the tetra-fluoroacetamide alcohol as a substantiallyexclusive product. Conversion of the tetra-fluoroacetamide alcohol tothe corresponding toluenesulfonate provides a material which is expectedto cyclize analogously to the tetra-toluenesulfonamide toluenesulfonateof the prior art. The cyclic tetra-amide product of the cyclization ofthe toluenesulfonate of tetra-fluoroacetamide alcohol, in methanolicsodium hydroxide at 15-25° C. for 1 hour, should affordnitro-benzyl-DOTA as a substantially exclusive product. As a result, theuse of trifluoracetamide protecting groups circumvents the difficultiesassociated with cleavage of the very stable toluenesulfonamideprotecting group, which involves heating with a large excess of sulfuricacid followed by neutralization with copious volumes of bariumhydroxide.

Alternatively, the problems in the prior art with respect to conversionof11-(p-nitrobenzyl)-N,N',N",N'",o-pentakis(tosyl-sulfonyl)-3,6,9,12-tetraazadodecanolto nitro-benzyl-DOTA may be overcome by the following procedure:

transient protection of the hydroxy group;

tosylation of the free amines;

deprotection of the transiently protected hydroxy group;

tosylation of the deprotected hydroxy group; and

an intramolecular tosylate cyclization reaction to form atwelve-membered DOTA ring. This procedure is analogous to the onedescribed in Example XVII with respect to the intermediate2-(p-nitrobenzyl)-N,N',N",N'",o-pentakis(tosyl-sulfonyl)-3,6,9,12-tetraazadodecanol.

Another alternative route to nitro-benzyl-DOTA is shown below. ##STR32##This alternative procedure involves the cyclizaton ofp-nitrophenylalanyltriglycine using a coupling agent, such asdiethylycyanophosphate, to give the cyclic tetraamide. Subsequent boranereduction provides 2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane, acommon precursor used in published routes to DOTA including the Renn andMeares article referenced above. This alternative procedure of thepresent invention offers a synthetic pathway that is considerablyshorter than the prior art Renn and Meares route, requiring two ratherthan four steps between p-nitrophenylalanyltriglycine to the tetraamine.The procedure of the present invention also avoids the use of tosylamino protecting groups, which were prepared in low yield and requiredstringent conditions for removal. Also, the procedure of the presentinvention poses advantages over the route published by Gansow et al.,U.S. Pat. No. 4,923,985, because the crucial cyclization step isintramolecular rather than intermolecular. Intramolecular reactionstypically proceed in higher yield and do not require high dilutiontechniques necessary for successful intermolecular reactions.

Additional routes to DOTA chelate compounds are set forth in ExampleXVII. These routes include the production of DOTA chelate compoundsemploying a phenylalanine or substituted phenylalanine startingmaterial. Preferable substituted phenylalanine starting materials aresubstituted at the para position and include NO₂ and NHR' wherein R' isan amino protecting group. Another preferred para substituent is NH--SO₂--2,3,6-methyl-4-methoxyphenyl. Preferred amino protecting groupsinclude toluenesulfonyl, isonicotinyl-carbamate and 9-fluorenylmethylcarbamate.

Radiolysis of Y-90 plays a role in the stability of Y-90-labeledcompounds. During radiolysis, Y-90, a high energy beta-emitter thatgives off a beta particle having a maximum energy of about 2.27 MeV,generates a greater number of oxidizing oxy-radicals (e.g., superoxideanion, hydroperoxy radical, hydrogen peroxide, and the like) perparticle emitted. Consequently, these active oxidants attack theY-90-chelate complex or other constituents of the complex resulting ininter- or intra-molecular decomposition. This phenomena has beenobserved with respect to strong beta-emitters, such as Cu-67, I-131,Re-186 and Re-188.

The DOTA-containing compounds discussed above incorporate a N₄macrocyclic ring structure. Studies discussed further below in ExampleXVIII have shown that a radioprotectant serves to stabilize Y-90-labeledDOTA-biotin compounds with respect to radiolysis. Preferredradioprotectants of the present invention are anti-oxidants (e.g.,either reduce the number or the activity of oxidizing radicals).

Exemplary radioprotectants that may be employed in the practice of thepresent invention are gentisic acid, ascorbic acid, nicotinic acid,ascorbyl palmitate, HOP(:O)H₂, monothioglycerol, sodium formaldehydesulfoxylate, Na₂ S₂ O₅, Na₂ S₂ O₃, ascorbate, SO₂, or a reducing agentcombined with BHA, BHT, Pr gallate or tocopherol and the like. Ascorbicacid is a preferred radioprotectant for use in the practice of thepresent invention.

The present invention also provides radiolabeled ligand moleculesbearing chemically modified ligand components. Whenbiotin-N-methyl-glycine-aminobenzyl-DOTA is oxidized with sodiumperiodate and the resultant oxidized product is labeled with Y-90, thiscompound exhibits the same HPLC retention time as the radiolysis productof the radiolabeled biotin-DOTA conjugate. NMR characterization of theoxidized biotin-N-methyl-glycine-aminobenzyl-DOTA is consistent withbiotin-sulfoxide formation. In view of these results, it appears thatradiolysis causes oxidation of the biotin heterocycle. The radiolysisand oxidized biotin products retain avidin binding capability whentested in vitro. See, for example, Example XVIII. Consequently, oxidizedbiotin-containing conjugates may be employed in the practice ofpretargeting embodiments of the present invention. This data alsoindicates that radiolysis of Y-90-radiolabeled biotin does not result inthe release of free Y-90.

The present invention also provides an article of manufacture whichincludes packaging material and an active agent-ligand or activeagent-anti-ligand conjugate, such as an Y-90-DOTA-biotin conjugate,contained within the packaging material, wherein the conjugate, uponadministration to a mammalian recipient, is capable of localizing at atarget site at which a previously administered complementaryligand/anti-ligand pair member has localized, and wherein the packagingmaterial includes a label that identifies the ligand or anti-ligandcomponent of the conjugate, identifies the active agent component of theconjugate and indicates an appropriate use of the conjugate in humanrecipients.

The packaging material indicates whether the conjugate is limited toinvestigational use or identifies an indication for which the conjugatehas been approved by the U.S. Food and Drug Administration or othersimilar regulatory body for use in humans. The packaging material mayalso include additional information including the amount of conjugate,the medium or environment in which the conjugate is dispersed, if any,lot number or other identifier, storage instructions, usageinstructions, a warning with respect to any restriction upon use of theconjugate, the name and address of the company preparing and/orpackaging the conjugate, and other information concerning the conjugate.

The active agent-ligand conjugate or active agent-anti-ligand conjugateis preferably contained within a vial which allows the conjugate to betransported prior to use. Such conjugate is preferably vialed in asterile, pyrogen-free environment. Alternatively, the conjugate may belyophilized prior to packaging. In this circumstance, instructions forpreparing the lyophilized conjugate for administration to a recipientmay be included on the label.

One component to be administered in a preferred two-step pretargetingprotocol is a targeting moiety-anti-ligand or a targeting moiety-ligandconjugate. In three-step pretargeting, a preferred component foradministration is a targeting moiety-ligand conjugate. A preferredtargeting moiety useful in these embodiments of the present invention isa monoclonal antibody. Protein-protein conjugations are generallyproblematic due to the formation of undesirable byproducts, includinghigh molecular weight and cross-linked species, however. A non-covalentsynthesis technique involving reaction of biotinylated antibody withstreptavidin has been reported to result in substantial byproductformation. Also, at least one of the four biotin binding sites on thestreptavidin is used to link the antibody and streptavidin, whileanother such binding site may be sterically unavailable for biotinbinding due to the configuration of the streptavidin-antibody conjugate.

Thus, covalent streptavidin-antibody conjugation is preferred, but highmolecular weight byproducts are often obtained. The degree ofcrosslinking and aggregate formation is dependent upon several factors,including the level of protein derivitization using heterobifunctionalcrosslinking reagents. Sheldon et al., Appl. Radiat. Isot. 43:1399-1402, 1992, discuss preparation of covalent thioether conjugates byreacting succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC)-derivitized antibody and iminothiolane-derivitized streptavidin.

Streptavidin-proteinaceous targeting moiety conjugates are preferablyprepared as described in Example XI below, with the preparationinvolving the steps of: preparation of SMCC-derivitized streptavidin;preparation of DTT-reduced proteinaceous targeting moiety; conjugationof the two prepared moieties; and purification of the monosubstitutedand/or disubstituted (with streptavidin) conjugate from crosslinked(antibody-streptavidin-antibody) and aggregate species. The purifiedfraction is preferably further characterized by one or more of thefollowing techniques: HPLC size exclusion, SDS-PAGE, immunoreactivity,biotin binding capacity and in vivo studies.

Alternatively, thioether conjugates useful in the practice of thepresent invention may be formed using other thiolating agents, such asSPDP, iminothiolane, SATA or the like, or other thio-reactiveheterobifunctional cross linkers, such asm-maleimidobenzoyl-N-hydroxysuccinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate or the like.

Streptavidin-proteinaceous targeting moiety conjugates of the presentinvention can also be formed by conjugation of a lysine epsilon aminogroup of one protein with a maleimide-derivitized form of the otherprotein. For example, at pH 8-10, lysine epsilon amino moieties reactwith protein maleimides, prepared, for instance, by treatment of theprotein with SMCC, to generate stable amine covalent conjugates. Inaddition, conjugates can be prepared by reaction of lysine epsilon aminomoieties of one protein with aldehyde functionalities of the otherprotein. The resultant imine bond is reducible to generate thecorresponding stable amine bond. Aldehyde functionalities may begenerated, for example, by oxidation of protein sugar residues or byreaction with aldehyde-containing heterobifunctional cross linkers.

Another method of forming streptavidin-targeting moiety conjugatesinvolves immobilized iminobiotin that binds SMCC-derivitizedstreptavidin. In this conjugation/purification method, the reversiblebinding character of iminobiotin (immobilized) to streptavidin isexploited to readily separate conjugate from the unreacted targetingmoiety. Iminobiotin binding can be reversed under conditions of lower pHand elevated ionic strength, e.g., NH₂ OAc, pH 4 (50 mM) with 0.5M NaCl.

For streptavidin, for example, the conjugation/purification proceeds asfollows:

SMCC-derivitized streptavidin is bound to immobilized iminobiotin(Pierce Chemical Co., St. Louis, Mo.), preferably in column format;

a molar excess (with respect to streptavidin) of DTT-reduced antibody(preferably free of reductant) is added to the nitrogen-purged,phosphate-buffered iminobiotin column wherein the SMCC-streptavidin isbound (DTT-reduced antibody will saturate the bound SMCC-streptavidin,and unbound reduced antibody passing through the column can be reused);

the column is washed free of excess antibody; and

a buffer that lowers the pH and increases ionic strength is added to thecolumn to elute streptavidin-antibody conjugate in pure form.

As indicated above, targeting moiety-mediated ligand-anti-ligandpretargeting involves the localization of either targeting moiety-ligandor targeting moiety-anti-ligand at target tissue. Often, peak uptake tosuch target tissue is achieved before the circulating level of targetingmoiety-containing conjugate in the blood is sufficiently low to permitthe attainment of an optimal target-to-non-target conjugate ratio. Toobviate this problem, two approaches are useful. The first approachallows the targeting moiety-containing conjugate to clear from the bloodby "natural" or endogenous clearance mechanisms. This method iscomplicated by variations in systemic clearance of proteins and byendogenous ligand or anti-ligand. For example, endogenous biotin mayinterfere with the preservation of biotin binding sites on astreptavidin-targeting moiety conjugate.

The second approach for improving targeting moiety-ligand or targetingmoiety-anti-ligand conjugate target-to-blood ratio "chases" theconjugate from the circulation through in vivo complexation of conjugatewith a molecule constituting or containing the complementary anti-ligandor ligand. When biotinylated antibodies are used as a ligand-targetingmoiety conjugate, for example, avidin forms relatively large aggregatedspecies upon complexation with the circulating biotinylated antibody,which aggregated species are rapidly cleared from the blood by the RESuptake. See, for example, U.S. Pat. No. 4,863,713. One problem with thismethod, however, is the potential for cross-linking and internalizingtumor-bound biotinylated antibody by avidin.

When avidin-targeting moiety conjugates are employed, poly-biotinylatedtransferrin has been used to form relatively large aggregated speciesthat are cleared by RES uptake. See, for example, Goodwin, J. Nucl. Med.33(10):1816-18, 1992). Poly-biotinylated transferrin also has thepotential for cross-linking and internalizing tumor-boundavidinylated-targeting moiety, however. In addition, both "chase"methodologies involve the prolonged presence of aggregated moieties ofintermediate, rather than large, size (which are not cleared as quicklyas large size particles by RES uptake), thereby resulting in serumretention of subsequently administered ligand-active agent oranti-ligand active agent. Such serum retention unfavorably impacts thetarget cell-to-blood targeting ratio.

The present invention provides clearing agents of protein andnon-protein composition having physical properties facilitating use forin vivo complexation and blood clearance of anti-ligand/ligand (e.g.,avidin/biotin)-targeting moiety (e.g., antibody) conjugates. Theseclearing agents are useful in improving the target:blood ratio oftargeting moiety conjugate. Other applications of these clearing agentsinclude lesional imaging or therapy involving blood clots and the like,employing antibody-active agent delivery modalities. For example,efficacious anti-clotting agent provides rapid target localization andhigh target:non-target targeting ratio. Active agents administered inpretargeting protocols of the present invention using efficient clearingagents are targeted in the desirable manner and are, therefore, usefulin the imaging/therapy of conditions such as pulmonary embolism and deepvein thrombosis.

Clearing agents useful in the practice of the present inventionpreferably exhibit one or more of the following characteristics:

rapid, efficient complexation with targeting moiety-ligand (oranti-ligand) conjugate in vivo;

rapid clearance from the blood of targeting moiety conjugate capable ofbinding a subsequently administered complementary anti-ligand or ligandcontaining molecule;

high capacity for clearing (or inactivating) large amounts of targetingmoiety conjugate; and

low immunogenicity.

Preferred clearing agents include hexose-based and non-hexose basedmoieties. Hexose-based clearing agents are molecules that have beenderivatized to incorporate one or more hexoses (six carbon sugarmoieties) recognized by Ashwell receptors or other receptors such as themannose/N-acetylglucosamine receptor which are associated withendothelial cells and/or Kupffer cells of the liver or the mannose6-phosphate receptor. Exemplary of such hexoses are galactose, mannose,mannose 6-phosphate, N-acetylglusosamine and the like. Other moietiesrecognized by Ashwell receptors, including glucose, N-galactosamine,N-acetylgalactosamine, thioglycosides of galactose and, generally,D-galactosides and glucosides or the like may also be used in thepractice of the present invention. Galactose is the prototypicalclearing agent hexose derivative for the purposes of this description.Galactose thioglycoside conjugation to a protein is preferablyaccomplished in accordance with the teachings of Lee et al.,"2-Imino-2-methoxyethyl 1-Thioglycosides: New Reagents for AttachingSugars to Proteins," Biochemistry, 15(18): 3956, 1976. Another usefulgalactose thioglycoside conjugation method is set forth in Drantz et al,"Attachment of Thioglycosides to Proteins: Enhancement of Liver MembraneBinding," Biochemistry, 15(18): 3963, 1976. Thus, galactose-based andnon-galactose based molecules are discussed below.

Protein-type galactose-based clearing agents include proteins havingendogenous exposed galactose residues or which have been derivatized toexpose or incorporate such galactose residues. Exposed galactoseresidues direct the clearing agent to rapid clearance by endocytosisinto the liver through specific receptors therefor (Ashwell receptors).These receptors bind the clearing agent, and induce endocytosis into thehepatocyte, leading to fusion with a lysosome and recycle of thereceptor back to the cell surface. This clearance mechanism ischaracterized by high efficiency, high capacity and rapid kinetics.

An exemplary clearing agent of the protein-based/galactose-bearingvariety is the asialoorosomucoid derivative of human alpha-1 acidglycoprotein (orosomucoid, molecular weight=41,000 Dal, isoelectricpoint=1.8-2.7). The rapid clearance from the blood of asialoorosomucoidhas been documented by Galli, et al., J. of Nucl. Med. Allied Sci.32(2): 110-16, 1988.

Treatment of orosomucoid with neuraminidase removes sialic acidresidues, thereby exposing galactose residues. Other such derivatizedclearing agents include, for example, galactosylated albumin,galactosylated-IgM, galactosylated-IgG, asialohaptoglobin, asialofetuin,asialoceruloplasmin and the like.

Human serum albumin (HSA), for example, may be employed in a clearingagent of the present invention as follows:

(Hexose)_(m) --Human Serum Albumin (HSA)--(Ligand), wherein n is aninteger from 1 to about 10 and m is an integer from 1 to about 25 andwherein the hexose is recognized by Ashwell receptors. In a preferredembodiment of the present invention the ligand is biotin and the hexoseis galactose. More preferably, HSA is derivatized with from 10-20galactose residues and 1-5 biotin residues. Still more preferably, HSAclearing agents of the present invention are derivatized with from about12 to about 15 galactoses and 3 biotins. Derivatization with bothgalactose and biotin are conducted in a manner sufficient to produceindividual clearing agent molecules with a range of biotinylation levelsthat averages a recited whole number, such as 1, biotin. Derivatizationwith 3 biotins, for example, produces a product mixture made up ofindividual clearing agent molecules, substantially all of which havingat least one biotin residue. Derivatization with 1 biotin produces aclearing agent product mixture, wherein a significant portion of theindividual molecules are not biotin derivatized. The whole numbers usedin this description refer to the average biotinylation of the clearingagents under discussion.

In addition, clearing agents based upon human proteins, especially humanserum proteins, such as, for example, orosomucoid and human serumalbumin, human IgG, human-anti-antibodies of IgG and IgM class and thelike, are less immunogenic upon administration into the serum of a humanrecipient. Another advantage of using asialoorosomucoid is that humanorosomucoid is commercially available from, for example, Sigma ChemicalCo, St. Louis, Mo.

One way to prevent clearing agent compromise of target-bound conjugatethrough direct complexation is through use of a clearing agent of a sizesufficient to render the clearing agent less capable of diffusion intothe extravascular space and binding to target-associated conjugate. Thisstrategy is useful alone or in combination with the aforementionedrecognition that exposed galactose residues direct rapid liver uptake.This size-exclusion strategy enhances the effectiveness ofnon-galactose-based clearing agents of the present invention. Thecombination (exposed galactose and size) strategy improves theeffectiveness of "protein-type" or "polymer-type" galactose-basedclearing agents.

Galactose-based clearing agents include galactosylated, biotinylatedproteins (to remove circulating streptavidin-targeting moietyconjugates, for example) of intermediate molecular weight (ranging fromabout 40,000 to about 200,000 Dal), such as biotinylatedasialoorosomucoid, galactosyl-biotinyl-human serum albumin or othergalactosylated and biotinylated derivatives of non-immunogenic solublenatural proteins, as well as biotin- and galactose-derivatizedpolyglutamate, polylysine, polyarginine, polyaspartate and the like.High molecular weight moieties (ranging from about 200,000 to about1,000,000 Dal) characterized by poor target access, includinggalactosyl-biotinyl-IgM or -IgG (approximately 150,000 Dal) molecules,as well as galactose- and biotin-derivatized transferrin conjugates ofhuman serum albumin, IgG and IgM molecules and the like, can also beused as clearing agents of the claimed invention. Chemically modifiedpolymers of intermediate or high molecular weight (ranging from about40,000 to about 1,000,000 Dal), such as galactose- andbiotin-derivatized dextran, hydroxypropylmethacrylamide polymers,polyvinylpyrrolidone-polystyrene copolymers, divinyl ether-maleic acidcopolymers, pyran copolymers, or PEG, also have utility as clearingagents in the practice of the present invention. In addition, rapidlyclearing biotinylated liposomes (high molecular weight moieties withpoor target access) can be derivatized with galactose and biotin toproduce clearing agents for use in the practice of the presentinvention.

A further class of clearing agents useful in the present inventioninvolve small molecules (ranging from about 500 to about 10,000 Dal)derivatized with galactose and biotin that are sufficiently polar to beconfined to the vascular space as an in vivo volume of distribution.More specifically, these agents exhibit a highly charged structure and,as a result, are not readily distributed into the extravascular volume,because they do not readily diffuse across the lipid membranes liningthe vasculature. Exemplary of such clearing agents are mono- orpoly-biotin-derivatized6,6'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalene disulfonic acid] tetrasodium salt,mono- or poly-biotinyl-galactose-derivatized polysulfateddextran-biotin, mono- or poly-biotinyl-galactose-derivatizeddextran-biotin and the like.

The galactose-exposed or -derivatized clearing agents are preferablycapable of (1) rapidly and efficiently complexing with the relevantligand- or anti-ligand-containing conjugates via ligand-anti-ligandaffinity; and (2) clearing such complexes from the blood via thegalactose receptor, a liver specific degradation system, as opposed toaggregating into complexes that are taken up by the generalized RESsystem, including the lung and spleen. Additionally, the rapid kineticsof galactose-mediated liver uptake, coupled with the affinity of theligand-anti-ligand interaction, allow the use of intermediate or evenlow molecular weight carriers.

Non-galactose residue-bearing moieties of low or intermediate molecularweight (ranging from about 40,000 to about 200,000 Dal) localized in theblood may equilibrate with the extravascular space and, therefore, binddirectly to target-associated conjugate, compromising targetlocalization. In addition, aggregation-mediated clearance mechanismsoperating through the RES system are accomplished using a largestoichiometric excess of clearing agent. In contrast, the rapid bloodclearance of galactose-based clearing agents used in the presentinvention prevents equilibration, and the high affinityligand-anti-ligand binding allows the use of low stoichiometric amountsof such galactose-based clearing agents. This feature further diminishesthe potential for galactose-based clearing agents to compromisetarget-associated conjugate, because the absolute amount of suchclearing agent administered is decreased.

Clearing agent evaluation experimentation involving galactose- andbiotin-derivatized clearing agents of the present invention is detailedin Examples XIII and XVI. Specific clearing agents of the presentinvention that were examined during the Example XVI experimentation are(1) asialoorosomucoid-biotin, (2) human serum albumin derivatized withgalactose and biotin, and (3) a 70,000 dalton molecular weight dextranderivatized with both biotin and galactose. The experimentation showedthat proteins and polymers are derivatizable to contain both galactoseand biotin and that the resultant derivatized molecule is effective inremoving circulating streptavidin-protein conjugate from the serum ofthe recipient. Biotin loading was varied to determine the effects onboth clearing the blood pool of circulating avidin-containing conjugateand the ability to deliver a subsequently administered biotinylatedisotope to a target site recognized by the streptavidin-containingconjugate. The effect of relative doses of the administered componentswith respect to clearing agent efficacy was also examined.

Protein-type and polymer-type non-galactose-based clearing agentsinclude the agents described above, absent galactose exposure orderivitization and the like. These clearing agents act through anaggregation-mediated RES mechanism. In these embodiments of the presentinvention, the clearing agent used will be selected on the basis of thetarget organ to which access of the clearing agent is to be excluded.For example, high molecular weight (ranging from about 200,000 to about1,000,000 Dal) clearing agents will be used when tumor targets or clottargets are involved.

Another class of clearing agents includes agents that do not removecirculating ligand or anti-ligand/targeting moiety conjugates, butinstead "inactivate" the circulating conjugates by blocking the relevantanti-ligand or ligand binding sites thereon. These "cap-type" clearingagents are preferably small (500 to 10,000 Dal) highly chargedmolecules, which exhibit physical characteristics that dictate a volumeof distribution equal to that of the plasma compartment (i.e., do notextravasate into the extravascular fluid volume). Exemplary cap-typeclearing agents are poly-biotin-derivatized6,6'-[(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis(azo)bis[4-amino-5-hydroxy-1,3-naphthalene disulfonic acid] tetrasodium salt,poly-biotinyl-derivatized polysulfated dextran-biotin, mono- orpoly-biotinyl-derivatized dextran-biotin and the like.

Cap-type clearing agents are derivatized with the relevant anti-ligandor ligand, and then administered to a recipient of previouslyadministered ligand/or anti-ligand/targeting moiety conjugate. Clearingagent-conjugate binding therefore diminishes the ability of circulatingconjugate to bind any subsequently administered active agent-ligand oractive agent-anti-ligand conjugate. The ablation of active agent bindingcapacity of the circulating conjugate increases the efficiency of activeagent delivery to the target, and increases the ratio of target-boundactive agent to circulating active agent by preventing the coupling oflong-circulating serum protein kinetics with the active agent. Also,confinement of the clearing agent to the plasma compartment preventscompromise of target-associated ligand or anti-ligand.

Clearing agents of the present invention may be administered in singleor multiple doses. A single dose of biotinylated clearing agent, forexample, produces a rapid decrease in the level of circulating targetingmoiety-streptavidin, followed by a small increase in that level,presumably caused, at least in part, by re-equilibration of targetingmoiety-streptavidin within the recipient's physiological compartments. Asecond or additional clearing agent doses may then be employed toprovide supplemental clearance of targeting moiety-streptavidin.Alternatively, clearing agent may be infused intravenously for a timeperiod sufficient to clear targeting moiety-streptavidin in a continuousmanner.

Other types of clearing agents and clearance systems are also useful inthe practice of the present invention to remove circulating targetingmoiety-ligand or -anti-ligand conjugate from the recipient'scirculation. Particulate-based clearing agents, for example, arediscussed in Example IX. In addition, extracorporeal clearance systemsare discussed in Example IX. In vivo clearance protocols employingarterially inserted proteinaceous or polymeric multiloop devices arealso described in Example IX.

One embodiment of the present invention in which rapid acting clearingagents are useful is in the delivery of Auger emitters, such as I-125,I-123, Er-165, Sb-119, Hg-197, Ru-97, Tl-201 and I-125 and Br-77, ornucleus-binding drugs to target cell nuclei. In these embodiments of thepresent invention, targeting moieties that localize to internalizingreceptors on target cell surfaces are employed to deliver a targetingmoiety-containing conjugate (i.e., a targeting moiety-anti-ligandconjugate in the preferred two-step protocol) to the target cellpopulation. Such internalizing receptors include EGF receptors,transferrin receptors, HER2 receptors, IL-2 receptors, otherinterleukins and cluster differentiation receptors, somatostatinreceptors, other peptide binding receptors and the like.

After the passage of a time period sufficient to achieve localization ofthe conjugate to target cells, but insufficient to induceinternalization of such targeted conjugates by those cells through areceptor-mediated event, a rapidly acting clearing agent isadministered. In a preferred two-step protocol, an activeagent-containing ligand or anti-ligand conjugate, such as a biotin-Augeremitter or a biotin-nucleus acting drug, is administered as soon as theclearing agent has been given an opportunity to complex with circulatingtargeting moiety-containing conjugate, with the time lag betweenclearing agent and active agent administration being less than about 24hours. In this manner, active agent is readily internalized throughtarget cell receptor-mediated internalization. While circulating Augeremitters are thought to be non-toxic, the rapid, specific targetingafforded by the pretargeting protocols of the present inventionincreases the potential of shorter half-life Auger emitters, such asI-123, which is available and capable of stable binding.

In order to more effectively deliver a therapeutic or diagnostic dose ofradiation to a target site, the radionuclide is preferably retained atthe tumor cell surface. Loss of targeted radiation occurs as aconsequence of metabolic degradation mediated by metabolically activetarget cell types, such as tumor or liver cells.

Preferable agents and protocols within the present invention aretherefore characterized by prolonged residence of radionuclide at thetarget cell site to which the radionuclide has localized and improvedradiation absorbed dose deposition at that target cell site, withdecreased targeted radioactivity loss resulting from metabolism.Radionuclides that are particularly amenable to the practice of thisaspect of the present invention are rhenium, iodine and like "non +3charged" radiometals which exist in chemical forms that easily crosscell membranes and are not, therefore, inherently retained by cells. Incontrast, radionuclides having a +3 charge, such as In-111, Y90, Lu-177and Ga-67, exhibit natural target cell retention as a result of theircontainment in high charge density chelates.

Evidence exists that streptavidin is resistant to metabolic degradation.Consequently, radionuclides bound directly or indirectly tostreptavidin, rather than, for example, directly to the targetingmoiety, are retained at target cell sites for extended periods of time.Streptavidin-associated radionuclides can be administered inpretargeting protocols intravenously, intraarterially or the like orinjected directly into lesions.

Monovalent antibody fragment-streptavidin conjugate may be used topretarget streptavidin, preferably in additional embodiments of thetwo-step aspect of the present invention. Exemplary monovalent antibodyfragments useful in these embodiments are Fv, Fab, Fab' and the like.Monovalent antibody fragments, typically exhibiting a molecular weightranging from about 25 kD (Fv) to about 50 kD (Fab, 9. Fab'), are smallerthan whole antibody and, therefore, are generally capable of greatertarget site penetration. Moreover, monovalent binding can result in lessbinding carrier restriction at the target surface (occurring during useof bivalent antibodies, which bind strongly and adhere to target cellsites thereby creating a barrier to further egress into sublayers oftarget tissue), thereby improving the homogeneity of targeting.

In addition, smaller molecules are more rapidly cleared from arecipient, thereby decreasing the immunogenicity of the administeredsmall molecule conjugate. A lower percentage of the administered dose ofa monovalent fragment conjugate localizes to target in comparison to awhole antibody conjugate. The decreased immunogenicity may permit agreater initial dose of the monovalent fragment conjugate to beadministered, however.

A multivalent, with respect to ligand, moiety is preferably thenadministered. This moiety also has one or more radionuclides associatedtherewith. As a result, the multivalent moiety serves as both a clearingagent for circulating anti-ligand-containing conjugate (throughcross-linking or aggregation of conjugate) and as a therapeutic agentwhen associated with target bound conjugate. In contrast to theinternalization caused by cross-linking described above, cross-linkingat the tumor cell surface stabilizes the monovalent fragment-anti-ligandmolecule and, therefore, enhances target retention, under appropriateconditions of antigen density at the target cell. In addition,monovalent antibody fragments generally do not internalize as dobivalent or whole antibodies. The difficulty in internalizing monovalentantibodies permits cross-linking by a monovalent moiety serves tostabilize the bound monovalent antibody through multipoint binding. Thistwo-step protocol of the present invention has greater flexibility withrespect to dosing, because the decreased fragment immunogenicity allowsmore streptavidin-containing conjugate, for example, to be administered,and the simultaneous clearance and therapeutic delivery removes thenecessity of a separate controlled clearing step.

Another embodiment of the pretargeting methodologies of the presentinvention involves the route of administration of the ligand- oranti-ligand-active agents. In these embodiments of the presentinvention, the active agent-ligand (e.g., radiolabeled biotin) or-anti-ligand is administered intraarterially using an artery supplyingtissue that contains the target. In the radiolabeled biotin example, thehigh extraction efficiency provided by avidin-biotin interactionfacilitates delivery of very high radioactivity levels to the targetcells, provided the radioactivity specific activity levels are high. Thelimit to the amount of radioactivity delivered therefore becomes thebiotin binding capacity at the target (i.e., the amount of antibody atthe target and the avidin equivalent attached thereto).

For these embodiments of the pretargeting methods of the presentinvention, particle emitting therapeutic radionuclides resulting fromtransmutation processes (without non-radioactive carrier forms present)are preferred. Exemplary radionuclides include Y-90, Re-188, At-211,Bi-212 and the like. Other reactor-produced radionuclides are useful inthe practice of these embodiments of the present invention, if they areable to bind in amounts delivering a therapeutically effective amount ofradiation to the target. A therapeutically effective amount of radiationranges from about 1500 to about 10,000 cGy depending upon severalfactors known to nuclear medicine practitioners.

Intraarterial administration pretargeting can be applied to targetspresent in organs or tissues for which supply arteries are accessible.Exemplary applications for intraarterial delivery aspects of thepretargeting methods of the present invention include treatment of livertumors through hepatic artery administration, brain primary tumors andmetastases through carotid artery administration, lung carcinomasthrough bronchial artery administration and kidney carcinomas throughrenal artery administration. Intraarterial administration pretargetingcan be conducted using chemotherapeutic drug, toxin and anti-tumoractive agents as discussed below. High potency drugs, lymphokines, suchas IL-2 and tumor necrosis factor, drug/lymphokine-carrier-biotinmolecules, biotinylated drugs/lymphokines, anddrug/lymphokine/toxin-loaded, biotin-derivitized liposomes are exemplaryof active agents and/or dosage forms useful for the delivery thereof inthe practice of this embodiment of the present invention.

In embodiments of the present invention employing radionuclidetherapeutic agents, the rapid clearance of nontargeted therapeutic agentdecreases the exposure of non-target organs, such as bone marrow, to thetherapeutic agent. Consequently, higher doses of radiation can beadministered absent dose limiting bone marrow toxicity. In addition,pretargeting methods of the present invention optionally includeadministration of short duration bone marrow protecting agents, such asWR 2721. As a result, even higher doses of radiation can be given,absent dose limiting bone marrow toxicity.

An additional aspect of the present invention is directed to the use oftargeting moieties that are monoclonal antibodies or fragments thereofthat localize to an antigen that is recognized by the antibody NR-LU-10.Such monoclonal antibodies or fragments may be murine or of othernon-human mammalian origin, chimeric, humanized or human.

NR-LU-10 is a 150 kilodalton molecular weight IgG2b monoclonal antibodythat recognizes an approximately 40 kilodalton glycoprotein antigenexpressed on most carcinomas. In vivo studies in mice using an antibodyspecific for the NR-LU-10 antigen revealed that such antibody was notrapidly internalized, which would have prevented localization of thesubsequently administered active-agent-containing conjugate to thetarget site.

NR-LU-10 is a well characterized pancarcinoma antibody that has beensafely administered to over 565 patients in human clinical trials. Thehybridoma secreting NR-LU-10 was developed by fusing mouse splenocytesimmunized with intact cells of a human small cell lung carcinoma withP3×63/Ag8UI murine myeloma cells. After establishing a seed lot, thehybridoma was grown via in vitro cell culture methods, purified andverified for purity and sterility.

Radioimmunoassays, immunoprecipitation and Fluorescence-Activated CellSorter (FACS) analysis were used to obtain reactivity profiles ofNR-LU-10. The NR-LU-10 target antigen was present on either fixedcultured cells or in detergent extracts of various types of cancercells. For example, the NR-LU-10 antigen is found in small cell lung,non-small cell lung, colon, breast, renal, ovarian, pancreatic, andother carcinoma tissues. Tumor reactivity of the NR-LU-10 antibody isset forth in Table A, while NR-LU-10 reactivity with normal tissues isset forth in Table B. The values in Table B are obtained as describedbelow. Positive NR-LU-10 tissue reactivity indicates NR-LU-10 antigenexpression by such tissues. The NR-LU-10 antigen has been furtherdescribed by Varki et al., "Antigens Associated with a Human LungAdenocarcinoma Defined by Monoclonal Antibodies," Cancer Research, 44:681-687, 1984, and Okabe et al., "Monoclonal Antibodies to SurfaceAntigens of Small Cell Carcinoma of the Lung," Cancer Research, 44:5273-5278, 1984.

The tissue specimens were scored in accordance with three reactivityparameters: (1) the intensity of the reaction; (2) the uniformity of thereaction within the cell type; and (3) the percentage of cells reactivewith the antibody. These three values are combined into a singleweighted comparative value between 0 and 500, with 500 being the mostintense reactivity. This comparative value facilitates comparison ofdifferent tissues. Table B includes a summary reactivity value, thenumber of tissue samples examined and the number of samples that reactedpositively with NR-LU-10.

Methods for preparing antibodies that bind to epitopes of the NR-LU-10antigen are described in U.S. Pat. No. 5,084,396. Briefly, suchantibodies may be prepared by the following procedure:

absorbing a first monoclonal antibody directed against a first epitopeof a polyvalent antigen onto an inert, insoluble matrix capable ofbinding immunoglobulin, thereby forming an immunosorbent;

combining the immunosorbent with an extract containing polyvalentNR-LU-10 antigen, forming an insolubilized immune complex wherein thefirst epitope is masked by the first monoclonal antibody;

immunizing an animal with the insolubilized immune complex;

fusing spleen cells from the immunized animal to myeloma cells to form ahybridoma capable of producing a second monoclonal antibody directedagainst a second epitope of the polyvalent antigen;

culturing the hybridoma to produce the second monoclonal antibody; and

collecting the second monoclonal antibody as a product of the hybridoma.

Consequently, monoclonal antibodies NR-LU-01, NR-LU-02 and NR-LU-03,prepared in accordance with the procedures described in theaforementioned patent, are exemplary targeting moieties useful in thisaspect of the present invention.

Additional antibodies reactive with the NR-LU-10 antigen may also beprepared by standard hybridoma production and screening techniques. Anyhybridoma clones so produced and identified may be further screened asdescribed above to verify antigen and tissue reactivity.

                                      TABLE A                                     __________________________________________________________________________    TUMOR REACTIVITY OF ANTIBODY                                                  Organ/Cell Type                                                                            #Pos/                                                                             Intensity.sup.a                                                                      Percent.sup.b                                                                         Uniformity.sup.c                                Tumor Exam Avg. Range Avg. Range Avg. Range                                 __________________________________________________________________________    Pancreas Carcinoma                                                                         6/6 3  3   100                                                                              100  2.3                                                                              2-3                                          Prostate Carcinoma 9/9 2.8 2-3 95 80-100 2 1-3                                Lung Adenocarcinoma 8/8 3 3 100 100 2.2 1-3                                   Lung Small Cell Carcinoma 2/2 3 3 100 100 2 2                                 Lung 8/8 2.3 2-3 73  5-100 1.8 1-3                                            Squamous Cell Carcinoma                                                       Renal Carcinoma 8/9 2.2 2-3 83 75-100 1 1                                     Breast Adenocarcinoma 23/23 2.9 2-3 97 75-100 2.8 1-3                         Colon Carcinoma 12/12 2.9 2-3 98 95-100 2.9 2-3                               Malignant Melanoma Ocular 0/2 0 0 0 0 0 0                                     Malignant Melanoma  0/11 0 0 0 0 0 0                                          Ovarian Carcinoma 35/35 2.9 2.3 200 100 2.2 1-3                               Undifferentiatad                                                              Carcinoma 1/1 2 2 90 90 2 2                                                   Osteosarcoma 1/1 2 2 20 20 1 1                                                Synovial Sarcoma 0/1 0 0 0 0 0 0                                              Lymphoma 0/2 0 0 0 0 0 0                                                      Liposarcoma 0/2 0 0 0 0 0 0                                                   Uterine Leiomyosarcoma 0/1 0 0 0 0 0 0                                      __________________________________________________________________________     .sup.a Rated from 0-3, with 3 representing highest intensity                  .sup.b Percentsge of cells stained within the examined tissue section.        .sup.c Rates from 0-3 with 3 representing highest uniformity.            

                  TABLE B                                                         ______________________________________                                        Organ/Cell Type # Pos/Exam Summary Reactivity                                 ______________________________________                                        Adenoid                                                                         Epithelium 3/3 433                                                            Lymphoid Follicle-Central 0/3 0                                               Lymphoid Follicle-Peripheral 0/3 0                                            Mucus Gland 2/2 400                                                           Adipose Tissue                                                                Fat Cells 0/3 0                                                               Adrenal                                                                       Zona Fasciculata Cortex 0/3 0                                                 Zona Glomerulosa Cortex 0/3 0                                                 Zona Reticularis Cortex 0/3 0                                                 Medulla 0/3 0                                                                 Aorta                                                                         Endothelium 0/3 0                                                             Elastic Interna 0/3 0                                                         Tunica Adventitia 0/3 0                                                       Tunica Media 0/3 0                                                            Brain-Cerebellum                                                              Axons, Myelinated 0/3 0                                                       Microglia 0/3 0                                                               Neurons 0/3 0                                                                 Purkenje's Cells 0/3 0                                                        Brain-Cerebrum                                                                Axons, Myelinated 0/3 0                                                       Microglia 0/3 0                                                               Neurons 0/3 0                                                                 Brain-Midbrain                                                                Axons, Myelinated 0/3 0                                                       Microglia 0/3 0                                                               Neurons 0/3 0                                                                 Colon                                                                         Mucosal Epithelium 3/3 500                                                    Muscularis Externa 0/3 0                                                      Muscularis Mucosa 0/3 0                                                       Nerve Ganglia 0/3 0                                                           Serosa 0/1 0                                                                  Duodenum                                                                      Mucosal Epithelium 3/3 500                                                    Muscularis Mucosa 0/3 0                                                       Epididymis                                                                    Epithelium 3/3 419                                                            Smooth Muscle 0/3 0                                                           Spermatoza 0/1 0                                                              Esophagus                                                                     Epithelium 3/3 86                                                             Mucosal Gland 2/2 450                                                         Smooth Muscla 0/3 0                                                           Gall Bladder                                                                  Mucosal Epithelium 0/3 467                                                    Smooth Muscle 0/3 0                                                           Heart                                                                         Myocardium 0/3 0                                                              Serosa 0/1 0                                                                  Ileum                                                                         Lymph Node 0/2 0                                                              Mucosal Epithelium 0/2 0                                                      Muscularis Externa 0/1 0                                                      Muscularis Mucosa 0/2 0                                                       Nerve Ganglia 0/1 0                                                           Serosa 0/1 0                                                                  Jejunum                                                                       Lymph Node 0/1 0                                                              Mucosal Epithelium 2/2 400                                                    Muscularis Externa 0/2 0                                                      Muscularis Mucosa 0/2 0                                                       Nerve Ganglia 0/2 0                                                           Serosa 0/1 0                                                                  Kidney                                                                        Collecting Tubules 2/3 160                                                    Distal Convoluted Tubules 3/3 500                                             Glomerular Epithelium 0/3 0                                                   Mesangial 0/3 0                                                               Proximal Convoluted Tubules 3/3 500                                           Liver                                                                         Bile Duct 3/3 500                                                             Central Lobular Hepatocyte 1/3 4                                              Periportal Hepatocyte 1/3 40                                                  Kupffer Cells 0/3 0                                                           Lung                                                                          Alveolar Macrophage 0/3 0                                                     Bronchial Epithelium 0/2 0                                                    Bronchial Smooth Muscle 0/2 0                                                 Pneumocyte Type I 3/3 354                                                     Pneumocyte Type II 3/3 387                                                    Lymph Node                                                                    Lymphoid Follicle-Central 0/3 0                                               Lymphoid Follicle-Peripheral 0/3 0                                            Mammary Gland                                                                 Aveolar Epithelium 3/3 500                                                    Duct Epithelium 3/3 500                                                       Myoepithelium 0/3 0                                                           Muscle Skeletal                                                               Muscle Fiber 0/3 0                                                            Nerve                                                                         Axon, Myelinated 0/2 0                                                        Endoneurium 0/2 0                                                             Neurolemma 0/2 0                                                              Neuron 0/2 0                                                                  Perineurium 0/2 0                                                             Ovary                                                                         Corpus Luteum 0/3 0                                                           Epithelium 1/1 270                                                            Granulosa 1/3 400                                                             Serosa 0/3 0                                                                  Theca 0/3 0                                                                   Oviduct                                                                       Epithelium 1/1 500                                                            Smooth Muscle 0/3 0                                                           Pancreas                                                                      Acinar Cell 3/3 500                                                           Duct Epithelium 3/3 500                                                       Islet Cell 3/3 500                                                            Peritoneum                                                                    Mesothelium 0/1 0                                                             Pituitary                                                                     Adenohypophysis 2/2 500                                                       Neurohypophysis 0/2 0                                                         Placenta                                                                      Trophoblasts 0/3 0                                                            Prostate                                                                      Concretions 0/3 0                                                             Glandular Epithelium 3/3 400                                                  Smooth Muscle 0/3 0                                                           Rectum                                                                        Lymph Node 0/2 0                                                              Mucosal Epithelium 0/2 0                                                      Muscularis Externa 0/1 0                                                      Muscularis Mucosa 0/3 0                                                       Nerve Ganglia 0/3 0                                                           Salivary Gland                                                                Acinar Epithelium 3/3 500                                                     Duct Epithelium 3/3 500                                                       Skin                                                                          Apocrine Glands 3/3 280                                                       Basal Layer 3/3 33                                                            Epithelium 1/3 10                                                             Follicle 1/1 190                                                              Stratum Corneum 0/3 0                                                         Spinal Cord                                                                   Axons, Myelinated 0/2 0                                                       Microglial 0/2 0                                                              Neurons 0/2 0                                                                 Spleen                                                                        Lymphoid Follicle-Central 0/3 0                                               Lymphoid Follicle-Peripheral 0/3 0                                            Trabecular Smooth Muscle 0/3 0                                                Stomach                                                                       Chief Cells 3/3 290                                                           Mucosal Epithelium 3/3 367                                                    Muscularis Mucosa/Externa 0/3 0                                               Parietal Cells 3/3 290                                                        Smooth Muscle 0/3 0                                                           Stromal Tissue                                                                Adipose  0/63 0                                                               Arteriolar Smooth Muscle  0/120 0                                             Endothelium  0/120 0                                                          Fibrous Connective Tissue  0/120 0                                            Macrophages  0/117 0                                                          Mast Cells/Eosinophils  0/86 0                                                Testis                                                                        Interstitial Cells 0/3 0                                                      Sertoli Cells 3/3 93                                                          Thymus                                                                        Hassal's Epithelium 3/3 147                                                   Hassal's Keratin 3/3 333                                                      Lymphoid Cortex 0/3 0                                                         Lymphoid Medulla 3/3 167                                                      Thyroid                                                                       C-cells 0/3 0                                                                 Colloid 0/3 0                                                                 Follicular Epithelium 3/3 500                                                 Tonsil                                                                        Epithelium 1/3 500                                                            Lymphoid Follicle-Central 0/3 0                                               Lymphoid Follicle-Peripheral 0/3 0                                            Mucus Gland 1/1 300                                                           Striated Muscle 0/3 0                                                         Umbilical cord                                                                Epithelium 0/3 0                                                              Urinary Bladder                                                               Muscosal Epithelium 3/3 433                                                   Serosa 0/1 0                                                                  Smooth Muscle 0/3 0                                                           Uterus                                                                        Endometrial Epithelium 3/3 500                                                Endometrial Glands 3/3 500                                                    Smooth Muscle 0/3 0                                                           Vagina/Cervix                                                                 Epithelial Glands 1/1 500                                                     Smooth Muscle 0/2 0                                                           Squamous Epithelium 1/1 200                                                 ______________________________________                                    

The invention is further described through presentation of the followingexamples. These examples are offered by way of illustration, and not byway of limitation.

EXAMPLE I Synthesis of a Chelate-Biotin Conjugate

A chelating compound that contains an N₃ S chelating core was attachedvia an amide linkage to biotin. Radiometal labeling of an exemplarychelate-biotin conjugate is illustrated below. ##STR33##

The spacer group "X" permits the biotin portion of the conjugate to besterically available for avidin binding. When "R¹ " is a carboxylic acidsubstituent (for instance, CH₂ COOH), the conjugate exhibits improvedwater solubility, and further directs in vivo excretion of theradiolabeled biotin conjugate toward renal rather than hepatobiliaryclearance.

Briefly, N-α-Cbz-N-Σ-t-BOC protected lysine was converted to thesuccinimidyl ester with NHS and DCC, and then condensed with asparticacid β-t-butyl ester. The resultant dipeptide was activated with NHS andDCC, and then condensed with glycine t-butyl ester. The Cbz group wasremoved by hydrogenolysis, and the amine was acylated usingtetrahydropyranyl mercaptoacetic acid succinimidyl ester, yieldingS-(tetrahydropyranyl)-mercaptoacetyl-lysine. Trifluoroacetic acidcleavage of the N-t-BOC group and t-butyl esters, followed bycondensation with LC-biotin-NHS ester provided (Σ-caproylamidebiotin)aspartyl glycine. This synthetic method is illustrated below.##STR34## ¹ H NMR: (CD₃ OD, 200 MHz Varian): 1.25-1.95 (m, 24H),2.15-2.25 (broad t, 4H), 2.65-3.05 (m, 4H), 3.30-3.45 (dd, 2H),3.50-3.65 (ddd, 2H), 3.95 (broad s, 2H), 4.00-4.15 (m, 1H), 4.25-4.35(m, 1H), 4.45-4.55 (m, 1H), 4.7-5.05 (m overlapping with HOD).

Elemental Analysis: C, H, N for C₃₅ H₅₇ N₇ O₁₁ S₂.H₂ O calculated:50.41, 7.13, 11.76 found: 50.13, 7.14, 11.40

EXAMPLE II Preparation of a Technetium or Rhenium RadiolabeledChelate-Biotin Conjugate

The chelate-biotin conjugate of Example I was radiolabeled with either^(99m) Tc pertechnetate or ¹⁸⁶ Re perrhenate. Briefly, ^(99m) Tcpertechnetate was reduced with stannous chloride in the presence ofsodium gluconate to form an intermediate Tc-gluconate complex. Thechelate-biotin conjugate of Example I was added and heated to 100° C.for 10 min at a pH of about 1.8 to about 3.3. The solution wasneutralized to a pH of about 6 to about 8, and yielded an N₃S-coordinated ^(99m) Tc-chelate-biotin conjugate. C-18 HPLC gradientelution using 5-60% acetonitrile in 1% acetic acid demonstrated twoanomers at 97% or greater radiochemical yield using δ (gamma ray)detection.

Alternatively, ¹⁸⁶ Re perrhenate was spiked with cold ammoniumperrhenate, reduced with stannous chloride, and complexed with citrate.The chelate-biotin conjugate of Example I was added and heated to 90° C.for 30 min at a pH of about 2 to 3. The solution was neutralized to a pHof about 6 to about 8, and yielded an N₃ S-coordinated ¹⁸⁶Re-chelate-biotin conjugate. C-18 HPLC gradient elution using 5-60%acetonitrile in 1% acetic acid resulted in radiochemical yields of85-90%. Subsequent purification over a C-18 reverse phase hydrophobiccolumn yielded material of 99% purity.

EXAMPLE III In Vitro Analysis of Radiolabeled Chelate-Biotin Conjugates

Both the ^(99m) Tc- and ¹⁸⁶ Re-chelate-biotin conjugates were evaluatedin vitro. When combined with excess avidin (about 100-fold molarexcess), 100% of both radiolabeled biotin conjugates complexed withavidin.

A ^(99m) Tc-biotin conjugate was subjected to various chemical challengeconditions. Briefly, ^(99m) Tc-chelate-biotin conjugates were combinedwith avidin and passed over a 5 cm size exclusion gel filtration column.The radiolabeled biotin-avidin complexes were subjected to variouschemical challenges (see Table 1), and the incubation mixtures werecentrifuged through a size exclusion filter. The percent ofradioactivity retained (indicating avidin-biotin-associated radiolabel)is presented in Table 1. Thus, upon chemical challenge, the radiometalremained associated with the macromolecular complex.

                  TABLE 1                                                         ______________________________________                                        Chemical Challenge of .sup.99m Tc-Chelate-                                      Biotin-Avidin Complexes                                                         Challenge             % Radioactivity Retained                            Medium      pH        1 h, 37° C.                                                                      18 h, RT                                      ______________________________________                                        PBS         7.2       99        99                                              Phosphate 8.0 97 97                                                           10 mM cysteine 8.0 92 95                                                      10 mM DTPA 8.0 99 98                                                          0.2 M carbonate 10.0 97 94                                                  ______________________________________                                    

In addition, each radiolabeled biotin conjugate was incubated at about50 μg/ml with serum; upon completion of the incubation, the samples weresubjected to instant thin layer chromatography (ITLC) in 80% methanol.Only 2-4% of the radioactivity remained at the origin (i.e., associatedwith protein); this percentage was unaffected by the addition ofexogenous biotin. When the samples were analyzed using size exclusionH-12 FPLC with 0.2M phosphate as mobile phase, no association ofradioactivity with serum macromolecules was observed.

Each radiolabeled biotin conjugate was further examined using acompetitive biotin binding assay. Briefly, solutions containing varyingratios of D-biotin to radiolabeled biotin conjugate were combined withlimiting avidin at a constant total biotin:avidin ratio. Avidin bindingof each radiolabeled biotin conjugate was determined by ITLC, and wascompared to the theoretical maximum stoichiometric binding (asdetermined by the HABA spectrophotometric assay of Green, Biochem. J.94:23c-24c, 1965). No significant difference in avidin binding wasobserved between each radiolabeled biotin conjugate and D-biotin.

EXAMPLE IV In Vivo Analysis of Radiolabeled Chelate-Biotin ConjugatesAdministered After Antibody Pretargeting

The ¹⁸⁶ Re-chelate-biotin conjugate of Example I was studied in ananimal model of a three-step antibody pretargeting protocol. Generally,this protocol involved: (i) prelocalization of biotinylated monoclonalantibody; (ii) administration of avidin for formation of a "sandwich" atthe target site and for clearance of residual circulating biotinylatedantibody; and (iii) administration of the ¹⁸⁶ Re-biotin conjugate fortarget site localization and rapid blood clearance.

A. Preparation and Characterization of Biotinylated Antibody

Biotinylated NR-LU-10 was prepared according to either of the followingprocedures. The first procedure involved derivitization of antibody vialysine ε-amino groups. NR-LU-10 was radioiodinated at tyrosines usingchloramine T and either ¹²⁵ I or ¹³¹ I sodium iodide. The radioiodinatedantibody (5-10 mg/ml) was then biotinylated using biotinamido caproateNHS ester in carbonate buffer, pH 8.5, containing 5% DMSO, according tothe scheme below. ##STR35##

The impact of lysine biotinylation on antibody immunoreactivity wasexamined. As the molar offering of biotin:antibody increased from 5:1 to40:1, biotin incorporation increased as expected (measured using theHABA assay and pronase-digested product) (Table 2, below). Percent ofbiotinylated antibody immunoreactivity as compared to native antibodywas assessed in a limiting antigen ELISA assay. The immunoreactivitypercentage dropped below 70% at a measured derivitization of 11.1:1;however, at this level of derivitization, no decrease was observed inantigen-positive cell binding (performed with LS-180 tumor cells atantigen excess). Subsequent experiments used antibody derivitized at abiotin:antibody ratio of 10:1.

                  TABLE 2                                                         ______________________________________                                        Effect of Lysine Biotinylation                                                  on Immunoreactivity                                                             Molar     Measured                                                          Offering Derivitization Immunoassessment (%)                                (Biotins/Ab)                                                                            (Biotins/Ab)  ELISA    Cell Binding                                 ______________________________________                                         5:1      3.4           86                                                      10:1 8.5 73 100                                                               13:1 11.1 69 102                                                              20:1 13.4 36 106                                                              40:1 23.1 27                                                                ______________________________________                                    

Alternatively, NR-LU-10 was biotinylated using thiol groups generated byreduction of cystines. Derivitization of thiol groups was hypothesizedto be less compromising to antibody immunoreactivity. NR-LU-10 wasradioiodinated using p-aryltin phenylate NHS ester (PIP-NHS) and either¹²⁵ I or ¹³¹ I sodium iodide. Radioiodinated NR-LU-10 was incubated with25 mM dithiothreitol and purified using size exclusion chromatography.The reduced antibody (containing free thiol groups) was then reactedwith a 10- to 100-fold molar excess of N-iodoacetyl-n'-biotinyl hexylenediamine in phosphate-buffered saline (PBS), pH 7.5, containing 5% DMSO(v/v).

                  TABLE 3                                                         ______________________________________                                        Effect of Thiol Biotinylation                                                   on Immunoreactivity                                                             Molar     Measured                                                          Offering Derivitization Immunoassessment (%)                                (Biotins/Ab)                                                                            (Biotins/Ab)  ELISA    Cell Binding                                 ______________________________________                                        10:1      4.7           114                                                     50:1 6.5 102 100                                                              100:1  6.1  95 100                                                          ______________________________________                                    

As shown in Table 3, at a 50:1 or greater biotin:antibody molaroffering, only 6 biotins per antibody were incorporated. No significantimpact on immunoreactivity was observed.

The lysine- and thiol-derivitized biotinylated antibodies ("antibody(lysine)" and "antibody (thiol)", respectively) were compared. Molecularsizing on size exclusion FPLC demonstrated that both biotinylationprotocols yielded monomolecular (monomeric) IgGs. Biotinylated antibody(lysine) had an apparent molecular weight of 160 kD, while biotinylatedantibody (thiol) had an apparent molecular weight of 180 kD. Reductionof endogenous sulfhydryls (i.e., disulfides) to thiol groups, followedby conjugation with biotin, may produce a somewhat unfoldedmacromolecule. If so, the antibody (thiol) may display a largerhydrodynamic radius and exhibit an apparent increase in molecular weightby chromatographic analysis. Both biotinylated antibody speciesexhibited 98% specific binding to immobilized avidin-agarose.

Further comparison of the biotinylated antibody species was performedusing non-reducing SDS-PAGE, using a 4% stacking gel and a 5% resolvinggel. Biotinylated samples were either radiolabeled or unlabeled and werecombined with either radiolabeled or unlabeled avidin or streptavidin.Samples were not boiled prior to SDS-PAGE analysis. The native antibodyand biotinylated antibody (lysine) showed similar migrations; thebiotinylated antibody (thiol) produced two species in the 50-75 kDrange. These species may represent two thiol-capped species. Under theseSDS-PAGE conditions, radiolabeled streptavidin migrates as a 60 kDtetramer. When 400 μg/ml radiolabeled streptavidin was combined with 50μg/ml biotinylated antibody (analogous to "sandwiching" conditions invivo), both antibody species formed large molecular weight complexes.However, only the biotinylated antibody (thiol)-streptavidin complexmoved from the stacking gel into the resolving gel, indicating adecreased molecular weight as compared to the biotinylated antibody(lysine)-streptavidin complex.

B. Blood Clearance of Biotinylated Antibody Species

Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was intravenouslyadministered to non-tumored nude mice at a dose of 100 μg. At 24 hpost-administration of radioiodinated biotinylated NR-LU-10, mice wereintravenously injected with either saline or 400 μg of avidin. Withsaline administration, blood clearances for both biotinylated antibodyspecies were biphasic and similar to the clearance of native NR-LU-10antibody.

In the animals that received avidin intravenously at 24 h, thebiotinylated antibody (lysine) was cleared (to a level of 5% of injecteddose) within 15 min of avidin administration (avidin:biotin=10:1). Withthe biotinylated antibody (thiol), avidin administration (10:1 or 25:1)reduced the circulating antibody level to about 35% of injected doseafter two hours. Residual radiolabeled antibody activity in thecirculation after avidin administration was examined in vitro usingimmobilized biotin. This analysis revealed that 85% of the biotinylatedantibody was complexed with avidin. These data suggest that thebiotinylated antibody (thiol)-avidin complexes that were formed wereinsufficiently crosslinked to be cleared by the RES.

Blood clearance and biodistribution studies of biotinylated antibody(lysine) 2 h post-avidin or post-saline administration were performed.Avidin administration significantly reduced the level of biotinylatedantibody in the blood (see FIG. 1), and increased the level ofbiotinylated antibody in the liver and spleen. Kidney levels ofbiotinylated antibody were similar.

EXAMPLE V In Vivo Characterization of ¹⁸⁶ Re-Chelate-Biotin ConjugatesIn a Three-Step Pretargeting Protocol

A ¹⁸⁶ Re-chelate-biotin conjugate of Example I (MW≈1000; specificactivity=1-2 mCi/mg) was examined in a three-step pretargeting protocolin an animal model. More specifically, 18-22 g female nude mice wereimplanted subcutaneously with LS-180 human colon tumor xenografts,yielding 100-200 mg tumors within 10 days of implantation.

NR-LU-10 antibody (MW≈150 kD) was radiolabeled with ¹²⁵ I/Chloramine Tand biotinylated via lysine residues (as described in Example IV.A,above). Avidin (MW≈66 kD) was radiolabeled with ¹³¹ I/PIP-NHS (asdescribed for radioiodination of NR-LU-10 in Example IV.A., above). Theexperimental protocol was as follows:

Group 1: Time 0, inject 100 μg ¹²⁵ I-labeled, biotinylated NR-LU-10

Time 24 h, inject 400 μg ¹³¹ I-labeled avidin

Time 26 h, inject 60 μg ¹⁸⁶ Re-chelate-biotin conjugate

Group 2: Time 0, inject 400 μg ¹³¹ I-labeled avidin

(control) Time 2 h, inject 60 μg ¹⁸⁶ Re-chelate-biotin conjugate

Group 3: Time 0, inject 60 μg ¹⁸⁶ Re-chelate-biotin conjugate

(control)

The three radiolabels employed in this protocol are capable of detectionin the presence of each other. It is also noteworthy that the sizes ofthe three elements involved are logarithmicallydifferent--antibody≅150,000; avidin≅66,000; and biotin≅1,000.Biodistribution analyses were performed at 2, 6, 24, 72 and 120 h afteradministration of the ¹⁸⁶ Re-chelate-biotin conjugate.

Certain preliminary studies were performed in the animal model prior toanalyzing the ¹⁸⁶ Re-chelate-biotin conjugate in a three-steppretargeting protocol. First, the effect of biotinylated antibody onblood clearance of avidin was examined. These experiments showed thatthe rate and extent of avidin clearance was similar in the presence orabsence of biotinylated antibody. Second, the effect of biotinylatedantibody and avidin on blood clearance of the ¹⁸⁶ Re-chelate-biotinconjugate was examined; blood clearance was similar in the presence orabsence of biotinylated antibody and avidin. Further, antibodyimmunoreactivity was found to be uncompromised by biotinylation at thelevel tested.

Third, tumor uptake of biotinylated antibody administered at time 0 orof avidin administered at time 24 h was examined. The results of thisexperimentation are shown in FIG. 1. At 25 h, about 350 pmol/gbiotinylated antibody was present at the tumor; at 32 h the level wasabout 300 pmol/g; at 48 h, about 200 pmol/g; and at 120 h, about 100pmol/g. Avidin uptake at the same time points was about 250, 150, 50 and0 pmol/g, respectively. From the same experiment, tumor to blood ratioswere determined for biotinylated antibody and for avidin. From 32 h to120 h, the ratios of tumor to blood were very similar.

Rapid and efficient removal of biotinylated antibody from the blood bycomplexation with avidin was observed. Within two hours of avidinadministration, a 10-fold reduction in blood pool antibody concentrationwas noted (FIG. 1), resulting in a sharp increase in tumor to bloodratios. Avidin is cleared rapidly, with greater than 90% of the injecteddose cleared from the blood within 1 hour after administration. TheRe-186-biotin chelate is also very rapidly cleared, with greater than99% of the injected dose cleared from the blood by 1 hour afteradministration.

The three-step pretargeting protocol (described for Group 1, above) wasthen examined. More specifically, tumor uptake of the ¹⁸⁶Re-chelate-biotin conjugate in the presence or absence of biotinylatedantibody and avidin was determined. In the absence of biotinylatedantibody and avidin, the ¹⁸⁶ Re-chelate-biotin conjugate displayed aslight peak 2 h post-injection, which was substantially cleared from thetumor by about 5 h. In contrast, at 2 h post-injection in the presenceof biotinylated antibody and avidin (specific), the ¹⁸⁶Re-chelate-biotin conjugate reached a peak in tumor approximately 7times greater than that observed in the absence of biotinylated antibodyand avidin. Further, the specifically bound ¹⁸⁶ Re-chelate-biotinconjugate was retained at the tumor at significant levels for more than50 h. Tumor to blood ratios determined in the same experiment increasedsignificantly over time (i.e., T:B=≈8 at 30 h; ≈15 at 100 h; ≈35 at 140h).

Tumor uptake of the ¹⁸⁶ Re-chelate-biotin conjugate has further beenshown to be dependent on the dose of biotinylated antibody administered.At 0 μg of biotinylated antibody, about 200 pmol/g of ¹⁸⁶Re-chelate-biotin conjugate was present at the tumor at 2 h afteradministration; at 50 μg antibody, about 500 pmol/g of ¹⁸⁶Re-chelate-biotin conjugate; and at 100 μg antibody, about 1,300 pmol/gof ¹⁸⁶ Re-chelate-biotin conjugate.

Rhenium tumor uptake via the three-step pretargeting protocol wascompared to tumor uptake of the same antibody radiolabeled throughchelate covalently attached to the antibody (conventional procedure).The results of this comparison are depicted in FIG. 2. Blood clearanceand tumor uptake were compared for the chelate directly labeled rheniumantibody conjugate and for the three-step pretargeted sandwich. Areasunder the curves (AUC) and the ratio of AUC_(tumor) /AUC_(blood) weredetermined. For the chelate directly labeled rhenium antibody conjugate,the ratio of AUC_(tumor) /AUC_(blood) =24055/10235 or 2.35; for thethree-step pretargeted sandwich, the ratio of AUC_(tumor) /AUC_(blood)=46764/6555 or 7.13.

Tumor uptake results are best taken in context with radioactivityexposure to the blood compartment, which directly correlates with bonemarrow exposure. Despite the fact that 100-fold more rhenium wasadministered to animals in the three-step protocol, the very rapidclearance of the small molecule (Re-186-biotin) from the blood minimizesthe exposure to Re-186 given in this manner. In the same matchedantibody dose format, direct labeled (conventional procedure) NR-LU-10whole antibody yielded greater exposure to rhenium than did the 100-foldhigher dose given in the three-step protocol. A clear increase in thetargeting ratio (tumor exposure to radioactivity:blood exposure toradioactivity--AUC_(tumor) :AUC_(blood)) was observed for three-steppretargeting (approximately 7:1) in comparison to the direct labeledantibody approach (approximately 2.4:1).

EXAMPLE VI Preparation of Chelate-Biotin Conjugates Having ImprovedBiodistribution Properties

The biodistribution of ¹¹¹ In-labeled-biotin derivatives varies greatlywith structural changes in the chelate and the conjugating group.Similar structural changes may affect the biodistribution of technetium-and rhenium-biotin conjugates. Accordingly, methods for preparingtechnetium- and rhenium-biotin conjugates having optimal clearance fromnormal tissue are advantageous.

A. Neutral MAMA Chelate/Conjugate

A neutral MAMA chelate-biotin conjugate is prepared according to thefollowing scheme. ##STR36## The resultant chelate-biotin conjugate showssuperior kidney excretion. Although the net overall charge of theconjugate is neutral, the polycarboxylate nature of the moleculegenerates regions of hydrophilicity and hydrophobicity. By altering thenumber and nature of the carboxylate groups within the conjugate,excretion may be shifted from kidney to gastrointestinal routes. Forinstance, neutral compounds are generally cleared by the kidneys;anionic compounds are generally cleared through the GI system.

B. Polylysine Derivitization

Conjugates containing polylysine may also exhibit beneficialbiodistribution properties. With whole antibodies, derivitization withpolylysine may skew the biodistribution of conjugate toward liveruptake. In contrast, derivitization of Fab fragments with polylysineresults in lower levels of both liver and kidney uptake; blood clearanceof these conjugates is similar to that of Fab covalently linked tochelate. An exemplary polylysine derivitized chelate-biotin conjugate isillustrated below. ##STR37## Inclusion of polylysine inradiometal-chelate-biotin conjugates is therefore useful for minimizingor eliminating RES sequestration while maintaining good liver and kidneyclearance of the conjugate. For improved renal excretion properties,polylysine derivatives are preferably succinylated followingbiotinylation. Polylysine derivatives offer the further advantages of:(1) increasing the specific activity of the radiometal-chelate-biotinconjugate; (2) permitting control of rate and route of blood clearanceby varying the molecular weight of the polylysine polymer; and (3)increasing the circulation half-life of the conjugate for optimal tumorinteraction.

Polylysine derivitization is accomplished by standard methodologies.Briefly, poly-L-lysine is acylated according to standard amino groupacylation procedures (aqueous bicarbonate buffer, pH 8, added biotin-NHSester, followed by chelate NHS ester). Alternative methodology involvesanhydrous conditions using nitrophenyl esters in DMSO and triethylamine. The resultant conjugates are characterized by UV and NMR spectra.

The number of biotins attached to polylysine is determined by the HABAassay. Spectrophotometric titration is used to assess the extent ofamino group derivitization. The radiometal-chelate-biotin conjugate ischaracterized by size exclusion.

C. Cleavable Linkage

Through insertion of a cleavable linker between the chelate and biotinportion of a radiometal-chelate-biotin conjugate, retention of theconjugate at the tumor relative to normal tissue may be enhanced. Morespecifically, linkers that are cleaved by enzymes present in normaltissue but deficient or absent in tumor tissue can increase tumorretention. As an example, the kidney has high levels of γ-glutamyltransferase; other normal tissues exhibit in vivo cleavage of γ-glutamylprodrugs. In contrast, tumors are generally deficient in enzymepeptidases. The glutamyl-linked biotin conjugate depicted below iscleaved in normal tissue and retained in the tumor. ##STR38## D. SerineLinker With O-Polar Substituent

Sugar substitution of N₃ S chelates renders such chelates water soluble.Sulfonates, which are fully ionized at physiological pH, improve watersolubility of the chelate-biotin conjugate depicted below. ##STR39##This compound is synthesized according to the standard reactionprocedures. Briefly, biocytin is condensed with N-t-BOC-(O-sulfonate orO-glucose) serine NHS ester to give N-t-BOC-(O-sulfonate or O-glucose)serine biocytinamide. Subsequent cleavage of the N-t-BOC group with TFAand condensation with ligand NHS ester in DMF with triethylamineprovides ligand-amidoserine(O-sulfonate or O-glucose)biocytinamide.

EXAMPLE VII Preparation and Characterization of PIP-RadioiodinatedBiotin

Radioiodinated biotin derivatives prepared by exposure of poly-L-lysineto excess NHS-LC-biotin and then to Bolton-Hunter N-hydroxysuccinimideesters in DMSO has been reported. After purification, this product wasradiolabeled by the iodogen method (see, for instance, Del Rosario etal., J. Nucl. Med. 32:5, 1991, 993 (abstr.)). Because of the highmolecular weight of the resultant radioiodinated biotin derivative, onlylimited characterization of product (i.e., radio-HPLC and binding toimmobilized streptavidin) was possible.

Preparation of radioiodinated biotin according to the present inventionprovides certain advantages. First, the radioiodobiotin derivative is alow molecular weight compound that is amenable to complete chemicalcharacterization. Second, the disclosed methods for preparation involvea single step and eliminate the need for a purification step.

Briefly, iodobenzamide derivatives corresponding to biocytin (R=COOH)and biotinamidopentylamine (R=H) were prepared according to thefollowing scheme. In this scheme, "X" may be any radiohalogen, including¹²⁵ I, ¹³¹ I, ¹²³ I, ²¹¹ At and the like. ##STR40## Preparation of 1 wasgenerally according to Wilbur et al., J. Nucl. Med. 30:216-26, 1989,using a tributyltin intermediate. Water soluble carbodiimide was used inthe above-depicted reaction, since the NHS ester 1 formed intractablemixtures with DCU. The NHS ester was not compatible with chromatography;it was insoluble in organic and aqueous solvents and did not react withbiocytin in DMF or in buffered aqueous acetonitrile. The reactionbetween 1 and biocytin or 5-(biotinamido) pentylamine was sensitive tobase. When the reaction of 1 and biocytin or the pentylamine wasperformed in the presence of triethylamine in hot DMSO, formation ofmore than one biotinylated product resulted. In contrast, the reactionwas extremely clean and complete when a suspension of 1 and biocytin (4mg/ml) or the pentylamine (4 mg/ml) was heated in DMSO at 117° C. forabout 5 to about 10 min. The resultant ¹²⁵ I-biotin derivatives wereobtained in 94% radiochemical yield. Optionally, the radioiodinatedproducts may be purified using C-18 HPLC and a reverse phase hydrophobiccolumn. Hereinafter, the resultant radioiodinated products 2 arereferred to as PIP-biocytin (R=COOH) and PIP-pentylamine (R=H).

Both iodobiotin derivatives 2 exhibited ≧95% binding to immobilizedavidin. Incubation of the products 2 with mouse serum resulted in noloss of the ability of 2 to bind to immobilized avidin. Biodistributionstudies of 2 in male BALB/c mice showed rapid clearance from the blood(similar to ¹⁸⁶ Re-chelate-biotin conjugates described above). Theradioiodobiotin 2 had decreased hepatobiliary excretion as compared tothe ¹⁸⁶ Re-chelate-biotin conjugate; urinary excretion was increased ascompared to the ¹⁸⁶ Re-chelate-biotin conjugate. Analysis of urinarymetabolites of 2 indicated deiodination and cleavage of the biotin amidebond; the metabolites showed no binding to immobilized avidin. Incontrast, metabolites of the ¹⁸⁶ Re-chelate-biotin conjugate appear tobe excreted in urine as intact biotin conjugates. Intestinal uptake of 2is <50% that of the ¹⁸⁶ Re-chelate-biotin conjugate. Thesebiodistribution properties of 2 provided enhanced whole body clearanceof radioisotope and indicate the advantageous use of 2 withinpretargeting protocols.

¹³¹ I-PIP-biocytin was evaluated in a two-step pretargeting procedure intumor-bearing mice. Briefly, female nude mice were injectedsubcutaneously with LS-180 tumor cells; after 7 d, the mice displayed50-100 mg tumor xenografts. At t=0, the mice were injected with 200 μgof NR-LU-10-avidin conjugate labeled with 125I using PIP-NHS (seeExample IV.A.). At t=36 h, the mice received 42 μg of 131I-PIP-biocytin.The data showed immediate, specific tumor localization, corresponding to≈1.5 ¹³¹ I-PIP-biocytin molecules per avidin molecule.

The described radiohalogenated biotin compounds are amenable to the sametypes of modifications described in Example VI above for ¹⁸⁶Re-chelate-biotin conjugates. In particular, the followingPIP-polylysine-biotin molecule is made by trace labeling polylysine with125I-PIP, followed by extensive biotinylation of the polylysine.##STR41## Assessment of ¹²⁵ I binding to immobilized avidin ensures thatall radioiodinated species also contain at least an equivalent ofbiotin.

EXAMPLE VIII Preparation of Biotinylated Antibody (Thiol) ThroughEndogenous Antibody Sulfhydryl Groups Or Sulfhydryl-Generating Compounds

Certain antibodies have available for reaction endogenous sulfhydrylgroups. If the antibody to be biotinylated contains endogenoussulfhydryl groups, such antibody is reacted withN-iodoacetyl-n'-biotinyl hexylene diamine (as described in ExampleIV.A., above). The availability of one or more endogenous sulfhydrylgroups obviates the need to expose the antibody to a reducing agent,such as DTT, which can have other detrimental effects on thebiotinylated antibody.

Alternatively, one or more sulfhydryl groups are attached to a targetingmoiety through the use of chemical compounds or linkers that contain aterminal sulfhydryl group. An exemplary compound for this purpose isiminothiolane. As with endogenous sulfhydryl groups (discussed above),the detrimental effects of reducing agents on antibody are therebyavoided.

EXAMPLE IX Two-Step Pretargeting Methodology That Does Not InduceInternalization

A NR-LU-13-avidin conjugate is prepared as follows. Initially, avidin isderivitized with N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). SMCC-derivedavidin is then incubated with NR-LU-13 in a 1:1 molar ratio at pH 8.5for 16 h. Unreacted NR-LU-13 and SMCC-derived avidin are removed fromthe mixture using preparative size exclusion HPLC. Two conjugates areobtained as products--the desired 1:1 NR-LU-13-avidin conjugate as themajor product; and an incompletely characterized component as the minorproduct.

A ^(99m) Tc-chelate-biotin conjugate is prepared as in Example II,above. The NR-LU-13-avidin conjugate is administered to a recipient andallowed to clear from the circulation. One of ordinary skill in the artof radioimmunoscintigraphy is readily able to determine the optimal timefor NR-LU-13-avidin conjugate tumor localization and clearance from thecirculation. At such time, the ^(99m) Tc-chelate-biotin conjugate isadministered to the recipient. Because the ^(99m) Tc-chelate-biotinconjugate has a molecular weight of ≈1,000, crosslinking ofNR-LU-13-avidin molecules on the surface of the tumor cells isdramatically reduced or eliminated. As a result, the ^(99m) Tcdiagnostic agent is retained at the tumor cell surface for an extendedperiod of time. Accordingly, detection of the diagnostic agent byimaging techniques is optimized; further, a lower dose of radioisotopeprovides an image comparable to that resulting from the typicalthree-step pretargeting protocol.

Optionally, clearance of NR-LU-13-avidin from the circulation may beaccelerated by plasmapheresis in combination with a biotin affinitycolumn. Through use of such column, circulating NR-LU-13-avidin will beretained extracorporeally, and the recipient's immune system exposure toa large, proteinaceous immunogen (i.e., avidin) is minimized.

Exemplary methodology for plasmapheresis/column purification useful inthe practice of the present invention is discussed in the context ofreducing radiolabeled antibody titer in imaging and in treating tumortarget sites in U.S. Pat. No. 5,078,673. Briefly, for the purposes ofthe present invention, an example of an extracorporeal clearancemethodology may include the following steps:

administering a ligand- or anti-ligand-targeting moiety conjugate to arecipient;

after a time sufficient for localization of the administered conjugateto the target site, withdrawing blood from the recipient by, forexample, plasmapheresis;

separating cellular element from said blood to produce a serum fractionand returning the cellular elements to the recipient; and

reducing the titer of the administered conjugate in the serum fractionto produce purified serum;

infusing the purified serum back into the recipient.

Clearance of NR-LU-13-avidin is also facilitated by administration of aparticulate-type clearing agent (e.g., a polymeric particle having aplurality of biotin molecules bound thereto). Such a particulateclearing agent preferably constitutes a biodegradable polymeric carrierhaving a plurality of biotin molecules bound thereto. Particulateclearing agents of the present invention exhibit the capability ofbinding to circulating administered conjugate and removing thatconjugate from the recipient. Particulate clearing agents of this aspectof the present invention may be of any configuration suitable for thispurpose. Preferred particulate clearing agents exhibit one or more ofthe following characteristics:

microparticulate (e.g., from about 0.5 micrometers to about 100micrometers in diameter, with from about 0.5 to about 2 micrometers morepreferred), free flowing powder structure;

biodegradable structure designed to biodegrade over a period of timebetween from about 3 to about 180 days, with from about 10 to about 21days more preferred, or non-biodegradable structure;

biocompatible with the recipients physiology over the course ofdistribution, metabolism and excretion of the clearing agent, morepreferably including biocompatible biodegradation products;

and capability to bind with one or more circulating conjugates tofacilitate the elimination or removal thereof from the recipient throughone or more binding moieties (preferably, the complementary member ofthe ligand/anti-ligand pair). The total molar binding capacity of theparticulate clearing agents depends upon the particle size selected andthe ligand or anti-ligand substitution ratio. The binding moieties arecapable of coupling to the surface structure of the particulate dosageform through covalent or non-covalent modalities as set forth herein toprovide accessible ligand or anti-ligand for binding to its previouslyadministered circulating binding pair member.

Preferable particulate clearing agents of the present invention arebiodegradable or non-biodegradable microparticulates. More preferably,the particulate clearing agents are formed of a polymer containingmatrix that biodegrades by random, nonenzymatic, hydrolytic scissioning.

Polymers derived from the condensation of alpha hydroxycarboxylic acidsand related lactones are more preferred for use in the presentinvention. A particularly preferred moiety is formed of a mixture ofthermoplastic polyesters (e.g., polylactide or polyglycolide) or acopolymer of lactide and glycolide components, such aspoly(lactide-co-glycolide). An exemplary structure, a randompoly(DL-lactide-co-glycolide), is shown below, with the values of x andy being manipulable by a practitioner in the art to achieve desirablemicroparticulate properties. ##STR42##

Other agents suitable for forming particulate clearing agents of thepresent invention include polyorthoesters and polyacetals (PolymerLetters, 18:293, 1980) and polyorthocarbonates (U.S. Pat. No. 4,093,709)and the like.

Preferred lactic acid/glycolic acid polymer containing matrixparticulates of the present invention are prepared by emulsion-basedprocesses, that constitute modified solvent extraction processes such asthose described by Cowsar et al., "Poly(Lactide-Co-Glycolide)Microcapsules for Controlled Release of Steroids," Methods Enzymology,112:101-116, 1985 (steroid entrapment in microparticulates); Eldridge etal., "Biodegradable and Biocompatible Poly(DL-Lactide-Co-Glycolide)Microspheres as an Adjuvant for Staphylococcal Enterotoxin B ToxoidWhich Enhances the Level of Toxin-Neutralizing Antibodies," Infectionand Immunity, 59:2978-2986, 1991 (toxoid entrapment); Cohen et al.,"Controlled Delivery Systems for Proteins Based on Poly(Lactic/GlycolicAcid) Microspheres," Pharmaceutical Research, 8(6):713-720, 1991 (enzymeentrapment); and Sanders et al., "Controlled Release of a LuteinizingHormone-Releasing Hormone Analogue from Poly(D,L-Lactide-Co-Glycolide)Microspheres," J. Pharmaceutical Science, 73(9):1294-1297, 1984 (peptideentrapment).

In general, the procedure for forming particulate clearing agents of thepresent invention involves dissolving the polymer in a halogenatedhydrocarbon solvent and adding an additional agent that acts as asolvent for the halogenated hydrocarbon solvent but not for the polymer.The polymer precipitates out from the polymer-halogenated hydrocarbonsolution. Following particulate formation, they are washed and hardenedwith an organic solvent. Water washing and aqueous non-ionic surfactantwashing steps follow, prior to drying at room temperature under vacuum.

For biocompatibility purposes, particulate clearing agents aresterilized prior to packaging, storage or administration. Sterilizationmay be conducted in any convenient manner therefor. For example, theparticulates can be irradiated with gamma radiation, provided thatexposure to such radiation does not adversely impact the structure orfunction of the binding moiety attached thereto. If the binding moietyis so adversely impacted, the particulate clearing agents can beproduced under sterile conditions.

The preferred lactide/glycolide structure is biocompatible with themammalian physiological environment. Also, these preferred sustainedrelease dosage forms have the advantage that biodegradation thereofforms lactic acid and glycolic acid, both normal metabolic products ofmammals.

Functional groups required for binding moiety--particulate bonding, areoptionally included in the particulate structure, along with thenon-degradable or biodegradable polymeric units. Functional groups thatare exploitable for this purpose include those that are reactive withligands or anti-ligands, such as carboxyl groups, amine groups,sulfhydryl groups and the like. Preferred binding enhancement moietiesinclude the terminal carboxyl groups of the preferred(lactide-glycolide) polymer containing matrix or the like. Apractitioner in the art is capable of selecting appropriate functionalgroups and monitoring conjugation reactions involving those functionalgroups.

Advantages garnered through the use of particulate clearing agents ofthe type described above are as follows:

particles in the "micron" size range localize in the RES and liver, withgalactose derivatization or charge modification enhancement methods forthis capability available, and, preferably, are designed to remain incirculation for a time sufficient to perform the clearance function;

the size of the particulates facilitates central vascular compartmentretention thereof, substantially precluding equilibration into theperipheral or extravascular compartment;

desired substituents for ligand or anti-ligand binding to theparticulates can be introduced into the polymeric structure;

ligand- or anti-ligand-particulate linkages having desired properties(e.g., serum biotinidase resistance thereby reducing the release ofbiotin metabolite from a particle-biotin clearing agent) and

multiple ligands or anti-ligands can be bound to the particles toachieve optimal cross-linking of circulating targeting agent-ligand or-anti-ligand conjugate and efficient clearance of cross-linked species.This advantage is best achieved when care is taken to preventparticulate aggregation both in storage and upon in vivo administration.

Clearance of NR-LU-13-avidin may also be accelerated by an arteriallyinserted proteinaceous or polymeric multiloop device. A catheter-likedevice, consisting of thin loops of synthetic polymer or protein fibersderivitized with biotin, is inserted into a major artery (e.g., femoralartery) to capture NR-LU-13-avidin. Since the total blood volume passesthrough a major artery every 70 seconds, the in situ clearing device iseffective to reduce circulating NR-LU-13-avidin within a short period oftime. This device offers the advantages that NR-LU-13-avidin is notprocessed through the RES; removal of NR-LU-13-avidin is controllableand measurable; and fresh devices with undiminished binding capacity areinsertable as necessary. This methodology is also useful withintraarterial administration embodiments of the present invention.

An alternative procedure for clearing NR-LU-13-avidin from thecirculation without induction of internalization involves administrationof biotinylated, high molecular weight molecules, such as liposomes, IgMand other molecules that are size excluded from ready permeability totumor sites. When such biotinylated, high molecular weight moleculesaggregate with NR-LU-13-avidin, the aggregated complexes are readilycleared from the circulation via the RES.

EXAMPLE X Enhancement of Therapeutic Agent Internalization ThroughAvidin Crosslinking

The ability of multivalent avidin to crosslink two or more biotinmolecules (or chelate-biotin conjugates) is advantageously used toimprove delivery of therapeutic agents. More specifically, avidincrosslinking induces internalization of crosslinked complexes at thetarget cell surface.

Biotinylated NR-CO-04 (lysine) is prepared according to the methodsdescribed in Example IV.A., above. Doxorubicin-avidin conjugates areprepared by standard conjugation chemistry. The biotinylated NR-CO-04 isadministered to a recipient and allowed to clear from the circulation.One of ordinary skill in the art of radioimmunotherapy is readily ableto determine the optimal time for biotinylated NR-CO-04 tumorlocalization and clearance from the circulation. At such time, thedoxorubicin-avidin conjugate is administered to the recipient. Theavidin portion of the doxorubicin-avidin conjugate crosslinks thebiotinylated NR-CO-04 on the cell surface, inducing internalization ofthe complex. Thus, doxorubicin is more efficiently delivered to thetarget cell.

In a first alternative protocol, a standard three-step pretargetingmethodology is used to enhance intracellular delivery of a drug to atumor target cell. By analogy to the description above, biotinylatedNR-LU-05 is administered, followed by avidin (for blood clearance and toform the middle layer of the sandwich at the target cell-boundbiotinylated antibody). Shortly thereafter, and prior to internalizationof the biotinylated NR-LU-05-avidin complex, a methotrexate-biotinconjugate is administered.

In a second alternative protocol, biotinylated NR-LU-05 is furthercovalently linked to methotrexate. Subsequent administration of avidininduces internalization of the complex and enhances intracellulardelivery of drug to the tumor target cell.

In a third alternative protocol, NR-CO-04-avidin is administered to arecipient and allowed to clear from the circulation and localize at thetarget site. Thereafter, a polybiotinylated species (such asbiotinylated poly-L-lysine, as in Example IV.B., above) is administered.In this protocol, the drug to be delivered may be covalently attached toeither the antibody-avidin component or to the polybiotinylated species.The polybiotinylated species induces internalization of the(drug)-antibody-avidin-polybiotin-(drug) complex.

EXAMPLE XI Targeting Moiety-Anti-Ligand Conjugate for Two-StepPretargeting In Vivo

A. Preparation of SMCC-derivitized stredtavidin.

31 mg (0.48 μmol) streptavidin was dissolved in 9.0 ml PBS to prepare afinal solution at 3.5 mg/ml. The pH of the solution was adjusted to 8.5by addition of 0.9 ml of 0.5M borate buffer, pH 8.5. A DMSO solution ofSMCC (3.5 mg/ml) was prepared, and 477 μl (4.8 μmol) of this solutionwas added dropwise to the vortexing protein solution. After 30 minutesof stirring, the solution was purified by G-25 (PD-10, Pharmacia,Piscataway, N.J.) column chromatography to remove unreacted orhydrolyzed SMCC. The purified SMCC-derivitized streptavidin was isolated(28 mg, 1.67 mg/ml).

B. Preparation of DTT-reduced NR-LU-10.

To 77 mg NR-LU-10 (0.42 μmol) in 15.0 ml PBS was added 1.5 ml of 0.5Mborate buffer, pH 8.5. A DTT solution, at 400 mg/ml (165 μl) was addedto the protein solution. After stirring at room temperature for 30minutes, the reduced antibody was purified by G-25 size exclusionchromatography. Purified DTT-reduced NR-LU-10 was obtained (74 mg, 2.17mg/ml).

C. Conjuration of SMCC-streptavidin to DTT-reduced NR-LU-10.

DTT-reduced NR-LU-10 (63 mg, 29 ml, 0.42 μmol) was diluted with 44.5 mlPBS. The solution of SMCC-streptavidin (28 mg, 17 ml, 0.42 μmol) wasadded rapidly to the stirring solution of NR-LU-10. Total proteinconcentration in the reaction mixture was 1.0 mg/ml. The progress of thereaction was monitored by HPLC (Zorbax® GF-250, available from MacMod).After approximately 45 minutes, the reaction was quenched by addingsolid sodium tetrathionate to a final concentration of 5 mM.

D. Purification of conjugate.

For small scale reactions, monosubstituted and/or disubstituted (withstreptavidin) conjugate was obtained using HPLC Zorbax (preparative)size exclusion chromatography. The desired monosubstituted and/ordisubstituted conjugate product eluted at 14.0-14.5 min (3.0 ml/min flowrate), while unreacted NR-LU-10 eluted at 14.5-15 min and unreactedderivitized streptavidin eluted at 19-20 min.

For larger scale conjugation reactions, monosubstituted and/ordisubstituted adduct is isolatable using DEAE ion exchangechromatography. After concentration of the crude conjugate mixture, freestreptavidin was removed therefrom by eluting the column with 2.5%xylitol in sodium borate buffer, pH 8.6. The bound unreacted antibodyand desired conjugate were then sequentially eluted from the columnusing an increasing salt gradient in 20 mM diethanolamine adjusted to pH8.6 with sodium hydroxide.

E. Characterization of Conjugate.

1. HPLC size exclusion was conducted as described above with respect tosmall scale purification.

2. SDS-PAGE analysis was performed using 5% polyacrylamide gels undernon-denaturing conditions. Conjugates to be evaluated were not boiled insample buffer containing SDS to avoid dissociation of streptavidin intoits 15 kD subunits. Two product bands were observed on the gel, whichcorrespond to the mono- and di- substituted conjugates.

3. Immunoreactivity was assessed, for example, by competitive bindingELISA as compared to free antibody. Values obtained were within 10% ofthose for the free antibody.

4. Biotin binding capacity was assessed, for example, by titrating aknown quantity of conjugate with p-[I-125]iodobenzoylbiocytin.Saturation of the biotin binding sites was observed upon addition of 4equivalences of the labeled biocytin.

5. In vivo studies are useful to characterize the reaction product,which studies include, for example, serum clearance profiles, ability ofthe conjugate to target antigen-positive tumors, tumor retention of theconjugate over time and the ability of a biotinylated molecule to bindstreptavidin conjugate at the tumor. These data facilitate determinationthat the synthesis resulted in the formation of a 1:1streptavidin-NR-LU-10 whole antibody conjugate that exhibits bloodclearance properties similar to native NR-LU-10 whole antibody, andtumor uptake and retention properties at least equal to native NR-LU-10.

For example, FIG. 3 depicts the tumor uptake profile of theNR-LU-10-streptavidin conjugate (LU-10-StrAv) in comparison to a controlprofile of native NR-LU-10 whole antibody. LU-10-StrAv was radiolabeledon the streptavidin component only, giving a clear indication thatLU-10-StrAv localizes to target cells as efficiently as NR-LU-10 wholeantibody itself.

EXAMPLE XII Two-Step Pretargeting In vivo

A ¹⁸⁶ Re-chelate-biotin conjugate (Re-BT) of Example I (MW≈1000;specific activity=1-2 mCi/mg) and a biotin-iodine-131 small molecule,PIP-Biocytin (PIP-BT, MW approximately equal to 602; specificactivity=0.5-1.0 mCi/mg), as discussed in Example VII above, wereexamined in a three-step pretargeting protocol in an animal model, asdescribed in Example V above. Like Re-BT, PIP-BT has the ability to bindwell to avidin and is rapidly cleared from the blood, with a serumhalf-life of about 5 minutes. Equivalent results were observed for bothmolecules in the two-step pretargeting experiments described herein.

NR-LU-10 antibody (MW≈150 kD) was conjugated to streptavidin (MW≈66 kD)(as described in Example XI above) and radiolabeled with ¹²⁵ I/PIP-NHS(as described for radioiodination of NR-LU-10 in Example IV.A., above).The experimental protocol was as follows:

Time 0 inject (i.v.) 200 μg NR-LU-10-StrAv conjugate;

Time 24-48 h inject (i.v.) 60-70 fold molar excess of radiolabeledbiotinyl molecule;

and perform biodistributions at 2, 6, 24, 72, 120 hours after injectionof radiolabeled biotinyl molecule

NR-LU-10-streptavidin has shown very consistent patterns of bloodclearance and tumor uptake in the LS-180 animal model. A representativeprofile is shown in FIG. 4. When either PIP-BT or Re-BT is administeredafter allowing the LU-10-StrAv conjugate to localize to target cellsites for at least 24 hours, the tumor uptake of therapeuticradionuclide is high in both absolute amount and rapidity. For PIP-BTadministered at 37 hours following LU-10-StrAv (I-125) administration,tumor uptake was above 500 pMOL/G at the 40 hour time point and peakedat about 700 pMOL/G at 45 hours post-LU-10-StrAv administration.

This almost instantaneous uptake of a small molecule therapeutic intotumor in stoichiometric amounts comparable to the antibody targetingmoiety facilitates utilization of the therapeutic radionuclide at itshighest specific activity. Also, the rapid clearance of radionuclidethat is not bound to LU-10-StrAv conjugate permits an increasedtargeting ratio (tumor:blood) by eliminating the slow tumor accretionphase observed with directly labeled antibody conjugates. The pattern ofradionuclide tumor retention is that of whole antibody, which is verypersistent.

Experimentation using the two-step pretargeting approach andprogressively lower molar doses of radiolabeled biotinyl molecule wasalso conducted. Uptake values of about 20% ID/G were achieved atno-carrier added (high specific activity) doses of radiolabeled biotinylmolecules. At less than saturating doses, circulating LU-10-StrAv wasobserved to bind significant amounts of administered radiolabeledbiotinyl molecule in the blood compartment.

EXAMPLE XIII Asialoorosomucoid Clearing Agent and Two-Step Pretargeting

In order to maximize the targeting ratio (tumor:blood), clearing agentswere sought that are capable of clearing the blood pool of targetingmoiety-anti-ligand conjugate (e.g., LU-10-StrAv), without compromisingthe ligand binding capacity thereof at the target sites. One such agent,biotinylated asialoorosomucoid, which employs the avidin-biotininteraction to conjugate to circulating LU-10-StrAv, was tested.

A. Derivitization of orosomucoid. 10 mg human orosomucoid (Sigma N-9885)was dissolved in 3.5 ml of pH 5.5 0.1M sodium acetate buffer containing160 mM NaCl. 70 μl of a 2% (w/v) CaCl solution in deionized (D.I.) waterwas added and 11 μl of neuraminidase (Sigma N-7885), 4.6 U/ml, wasadded. The mixture was incubated at 37° C. for 2 hours, and the entiresample was exchanged over a Centricon-10® ultrafiltration device(available from Amicon, Danvers, Mass.) with 2 volumes of PBS. Theasialoorosomucoid and orosomucoid starting material were radiolabeledwith I-125 using PIP technology, as described in Example IV above.

The two radiolabeled preparations were injected i.v. into female BALB/cmice (20-25 g), and blood clearance was assessed by serial retro-orbitaleye bleeding of each group of three mice at 5, 10, 15 and 30 minutes, aswell as at 1, 2 and 4 hours post-administration. The results of thisexperiment are shown in FIG. 5, with asialoorosomucoid clearing morerapidly than its orosomucoid counterpart.

In addition, two animals receiving each compound were sacrificed at 5minutes post-administration and limited biodistributions were performed.These results are shown in FIG. 6. The most striking aspects of thesedata are the differences in blood levels (78% for orosomucoid and 0.4%for asialoorosomucoid) and the specificity of uptake ofasialoorosomucoid in the liver (86%), as opposed to other tissues.

B. Biotinylation of asialoorosomucoid clearing agent and orosomucoidcontrol. 100 μl of 0.2M sodium carbonate buffer, pH 9.2, was added to 2mg (in 1.00 ml PBS) of PIP-125-labeled orosomucoid and to 2 mgPIP-125-labeled asialoorosomucoid. 60 μl of a 1.85 mg/ml solution ofNHS-amino caproate biotin in DMSO was then added to each compound. Thereaction mixtures were vortexed and allowed to sit at room temperaturefor 45 minutes. The material was purified by size exclusion columnchromatography (PD-10, Pharmacia) and eluted with PBS. 1.2 ml fractionswere taken, with fractions 4 and 5 containing the majority of theapplied radioactivity (>95%). Streptavidin-agarose beads (Sigma S-1638)or -pellets were washed with PBS, and 20 μg of each biotinylated,radiolabeled protein was added to 400 μl of beads and 400 μl of PBS,vortexed for 20 seconds and centrifuged at 14,000 rpm for 5 minutes. Thesupernatant was removed and the pellets were washed with 400 μl PBS.This wash procedure was repeated twice more, and the combinedsupernatants were assayed by placing them in a dosimeter versus theirrespective pellets. The values are shown below in Table 4.

                  TABLE 4                                                         ______________________________________                                        Compound         Supernatant  Pellet                                          ______________________________________                                        orosomucoid      90%          10%                                               biotin-oroso 7.7% 92.%                                                        asialoorosomucoid 92% 8.0%                                                    biotin-asialo 10% 90%                                                       ______________________________________                                    

C. Protein-Streptavidin Binding in vivo. Biotin-asialoorosomucoid wasevaluated for the ability to couple with circulating LU-10-StrAvconjugate in vivo and to remove it from the blood. Female BALB/c mice(20-25 g) were injected i.v. with 200 μg LU-10-StrAv conjugate. Clearingagent (200 μl PBS--group 1; 400 μg non-biotinylatedasialoorosomucoid--group 2; 400 μg biotinylated asialoorosomucoid--group3; and 200 μg biotinylated asialoorosomucoid--group 4) was administeredat 25 hours following conjugate administration. A fifth group receivedPIP-I-131-LU-10-StrAv conjugate which had been saturated prior toinjection with biotin--group 5. The 400 μg dose constituted a 10:1 molarexcess of clearing agent over the initial dose of LU-10-StrAv conjugate,while the 200 μg dose constituted a 5:1 molar excess. The saturatedPIP-I-131-LU-10-StrAv conjugate was produced by addition of a 10-foldmolar excess of D-biotin to 2 mg of LU-10-StrAv followed by sizeexclusion purification on a G-25 PD-10 column.

Three mice from each group were serially bled, as described above, at0.17, 1, 4 and 25 hours (pre-injection of clearing agent), as well as at27, 28, 47, 70 and 90 hours. Two additional animals from each group weresacrificed at 2 hours post-clearing agent administration and limitedbiodistributions were performed.

The blood clearance data are shown in FIG. 7. These data indicate thatcirculating LU-10-StrAv radioactivity in groups 3 and 4 was rapidly andsignificantly reduced, in comparison to those values obtained in thecontrol groups 1, 2 and 5. Absolute reduction in circulatingantibody-streptavidin conjugate was approximately 75% when compared tocontrols.

Biodistribution data are shown in tabular form in FIG. 8. Thebiodistribution data show reduced levels of conjugate for groups 3 and 4in all tissues except the liver, kidney and intestine, which isconsistent with the processing and excretion of radiolabel associatedwith the conjugate after complexation with biotinylatedasialoorosomucoid.

Furthermore, residual circulating conjugate was obtained from serumsamples by cardiac puncture (with the assays conducted in serum+PBS) andanalyzed for the ability to bind biotin (immobilized biotin on agarosebeads), an indicator of functional streptavidin remaining in the serum.Group 1 animal serum showed conjugate radiolabel bound about 80% toimmobilized biotin. Correcting the residual circulating radiolabelvalues by multiplying the remaining percent injected dose (at 2 hoursafter clearing agent administration) by the remaining percent able tobind immobilize biotin (the amount of remaining functional conjugate)leads to the graph shown in FIG. 9. Administration of 200 μgbiotinylated asialoorosomucoid resulted in a 50-fold reduction in serumbiotin-binding capacity and, in preliminary studies in tumored animals,has not exhibited cross-linking and removal of prelocalized LU-10-StrAvconjugate from the tumor. Removal of circulating targetingmoiety-anti-ligand without diminishing biotin-binding capacity at targetcell sites, coupled with an increased radiation dose to the tumorresulting from an increase in the amount of targeting moiety-anti-ligandadministered, results in both increased absolute rad dose to tumor anddiminished toxicity to non-tumor cells, compared to what is currentlyachievable using conventional radioimmunotherapy.

A subsequent experiment was executed to evaluate lower doses ofasialoorosomucoid-biotin. In the same animal model, doses of 50, 20 and10 μg asialoorosomucoid-biotin were injected at 24 hours followingadministration of the LU-10-StrAv conjugate. Data from animals seriallybled are shown in FIG. 10, and data from animals sacrificed two hoursafter clearing agent administration are shown in FIGS. 11A (bloodclearance) and 11B (serum biotin-binding), respectively. Doses of 50 and20 μg asialoorosomucoid-biotin effectively reduced circulatingLU-10-StrAv conjugate levels by about 65% (FIG. 11A) and, aftercorrection for binding to immobilized biotin, left only 3% of theinjected dose in circulation that possessed biotin-binding capacity,compared with about 25% of the injected dose in control animals (FIG.11B). Even at low doses (approaching 1:1 stoichiometry with circulatingLU-10-StrAv conjugate), asialoorosomucoid-biotin was highly effective atreducing blood levels of circulating streptavidin-containing conjugateby an in vivo complexation that was dependent upon biotin-avidininteraction.

EXAMPLE XIV Tumor Uptake of PIP-Biocytin

PIP-Biocytin, as prepared and described in Example VII above, was testedto determine the fate thereof in vivo. The following data are based onexperimentation with tumored nude mice (100 mg LS-180 tumor xenograftsimplanted subcutaneously 7 days prior to study) that received, at time0, 200 μg of I-125 labeled NR-LU-10-Streptavidin conjugate (950 pmol),as discussed in Example XI above. At 24 hours, the mice received an i.v.injection of PIP-I-131-biocytin (40 μCi) and an amount of cold carrierPIP-I-127 biocytin corresponding to doses of 42 μg (69,767 pmol), 21 μg(34,884 pmol), 5.7 μg (9468 pmol), 2.85 μg (4734 pmol) or 0.5 μg (830pmol). Tumors were excised and counted for radioactivity 4 hours afterPIP-biocytin injection.

The three highest doses produced PIP-biocytin tumor localizations ofabout 600 pmol/g. Histology conducted on tissues receiving the twohighest doses indicated that saturation of tumor-bound streptavidin wasachieved. Equivalent tumor localization observed at the 5.7 μg dose isindicative of streptavidin saturation as well. In contrast, the twolowest doses produced lower absolute tumor localization of PIP-biocytin,despite equivalent localization of NR-LU-10-Streptavidin conjugate(tumors in all groups averaged about 40% ID/G for the conjugate).

The lowest dose group (0.5 μg) exhibited high efficiency tumor deliveryof PIP-I-131-biocytin, which efficiency increased over time. A peakuptake of 85.0% ID/G was observed at the 120 hour time point (96 hoursafter administration of PIP-biocytin). Also, the absolute amount ofPIP-biocytin, in terms of % ID, showed a continual increase in the tumorover all of the sampled time points. The decrease in uptake on a % ID/Gbasis at the 168 hour time point resulted from significant growth of thetumors between the 120 and 168 hour time points.

In addition, the co-localization of NR-LU-10-Streptavidin conjugate(LU-10-StrAv) and the subsequently administered PIP-Biocytin at the sametumors over time was examined. The localization of radioactivity attumors by PIP-biocytin exhibited a pattern of uptake and retention thatdiffered from that of the antibody-streptavidin conjugate (LU-10-StrAv).LU-10-StrAv exhibited a characteristic tumor uptake pattern that isequivalent to historical studies of native NR-LU-10 antibody, reaching apeak value of 40% ID/G between 24 and 48 hours after administration. Incontrast, the PIP-Biocytin exhibited an initial rapid accretion in thetumor, reaching levels greater than those of LU-10-StrAv by 24 hoursafter PIP-Biocytin administration. Moreover, the localization ofPIP-Biocytin continued to increase out to 96 hours, when theconcentration of radioactivity associated with the conjugate has begunto decrease. The slightly greater amounts of circulating PIP-Biocytincompared to LU-10-StrAv at these time points appeared insufficient toaccount for this phenomenon.

The ratio of PIP-Biocytin to LU-10-StrAv in the tumor increasedcontinually during the experiment, while the ratio in the blooddecreased continually. This observation is consistent with a processinvolving continual binding of targeting moiety-containing conjugate(with PIP-Biocytin bound to it) from the blood to the tumor, withsubsequent differential processing of the PIP-Biocytin and theconjugate. Since radiolabel associated with the streptavidin conjugatecomponent (compared to radiolabel associated with the targeting moiety)has shown increased retention in organs of metabolic processing,PIP-Biocytin associated with the streptavidin appears to be selectivelyretained by the tumor cells. Because radiolabel is retained at targetcell sites, a greater accumulation of radioactivity at those sitesresults.

The AUC_(tumor) /AUC_(blood) for PIP-Biocytin is over twice that of theconjugate (4.27 compared to 1.95, where AUC means "area under thecurve"). Further, the absolute AUC_(tumor) for PIP-Biocytin is nearlytwice that of the conjugate (9220 compared to 4629). Consequently, anincrease in radiation dose to tumor was achieved.

EXAMPLE XV Synthesis of DOTA-Biotin conjugates

A. Synthesis of Nitro-Benzvl-DOTA.

The synthesis of aminobenzyl-DOTA was conducted substantially inaccordance with the procedure of McMurry et al., Bioconjugate Chem., 3:108-117, 1992. The critical step in the prior art synthesis is theintermolecular cyclization between disuccinimidylN-(tert-butoxycarbonyl)iminodiacetate and N-(2-aminoethyl)-4-nitrophenylalaninamide to prepare1-(tert-butoxycarbonyl)-5-(4-nitrobenzyl)-3,6,11-trioxo-1,4,7,10-tetraazacyclododecane.In other words, the critical step is the intermolecular cyclizationbetween the bis-NHS ester and the diamine to give the cyclized dodecane.McMurry et al. conducted the cyclization step on a 140 mmol scale,dissolving each of the reagents in 100 ml DMF and adding via a syringepump over 48 hours to a reaction pot containing 4 liters dioxane.

A 5× scale-up of the McMurry et al. procedure was not practical in termsof reaction volume, addition rate and reaction time. Process chemistrystudies revealed that the reaction addition rate could be substantiallyincreased and that the solvent volume could be greatly reduced, whilestill obtaining a similar yield of the desired cyclization product.Consequently on a 30 mmol scale, each of the reagents was dissolved in500 ml DMF and added via addition funnel over 27 hours to a reaction potcontaining 3 liters dioxane. The addition rate of the method employedinvolved a 5.18 mmol/hour addition rate and a 0.047M reactionconcentration.

B. Synthesis of a D-alanine-linked conjugate with a preserved biotincarboxy moiety.

A reaction scheme to form a compound of the following formula isdiscussed below. ##STR43##

The D-alanine-linked conjugate was prepared by first coupling D-alanine(Sigma Chemical Co.) to biotin-NHS ester. The resultantbiotinyl-D-alanine was then activated with1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) andN-hydroxysuccinimide (NHS). This NHS ester was reacted in situ withDOTA-aniline to give the desired product which was purified bypreparative HPLC.

More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent),triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) washeated at 110° C. for 30 minutes. The solution was cooled to 23° C. andevaporated. The product solid was acidified with glacial acetic acid andevaporated again. The product biotinyl-D-alanine, a white solid, wassuspended in 40 ml of water to remove excess unreacted D-alanine, andcollected by filtration. Biotinyl-D-alanine was obtained as a whitesolid (130 mg, 0.41 mmol) in 47% yield.

NHS (10 mg, 0.08 mmol) and EDCI (15 mg, 0.07 mmol) were added to asolution of biotinyl-D-alanine (27 mg, 0.08 mmol) in DMF (1 ml). Thesolution was stirred at 23° C. for 60 hours, at which time TLC analysisindicated conversion of the carboxyl group to the N-hydroxy succinimidylester. Pyridine (0.8 ml) was added followed by DOTA-aniline (20 mg, 0.04mmol). The mixture was heated momentarily at approximately 100° C., thencooled to 23° C. and evaporated. The product,DOTA-aniline-D-alanyl-biotinamide was purified by preparative HPLC.

C. Synthesis of N-hydroxvethyl-linked conjugate.

Iminodiacetic acid dimethyl ester is condensed with biotin-NHS-ester togive biotinyl dimethyl iminodiacetate. Hydrolysis with one equivalent ofsodium hydroxide provides the monomethyl ester after purification fromunder and over hydrolysis products. Reduction of the carboxyl group withborane provides the hydroxyethyl amide. The hydroxyl group is protectedwith t-butyl-dimethyl-silylchloride. The methyl ester is hydrolysed,activated with EDCI and condensed with DOTA-aniline to form the finalproduct conjugate.

D. Synthesis of N-Me-LC-DOTA-biotin.

A reaction scheme is shown below. ##STR44##

Esterification of 6-aminocaproic acid (Sigma Chemical Co.) was carriedout with methanolic HCl. Trifluoroacetylation of the amino group usingtrifluoroacetic anhydride gave N-6-(methylcaproyl) trifluoroacetamide.The amide nitrogen was methylated using sodium hydride and iodomethanein tetrahydrofuran. The trifluoroacetyl protecting group was cleaved inacidic methanol to give methyl 6-methylamino-caproate hydrochloride. Theamine was condensed with biotin-NHS ester to give methylN-methyl-caproylamido-biotin. Saponification afforded the correspondingacid which was activated with EDCI and NHS and, in situ, condensed withDOTA-aniline to give DOTA-benzylamido-N-methyl-caproylamido-biotin.

1. Preparation of methyl 6-aminocaproate hydrochloride. Hydrogenchloride (gas) was added to a solution of 20.0 g (152 mmol) of6-aminocaproic acid in 250 ml of methanol via rapid bubbling for 2-3minutes. The mixture was stirred at 15-25° C. for 3 hours and thenconcentrated to afford 27.5 g of the product as a white solid (99%):

H-NMR (DMSO) 9.35 (1 H, broad t), 3.57 (3H, s), 3.14 (2H, quartet), 2.28(2H, t), 1.48 (4H, multiplet), and 1.23 ppm (2H, multiplet).

2. Preparation of N-6-(methylcaproyl)-trifluoroacetamide. To a solutionof 20.0 g (110 mmol) of methyl 6-aminocaproate hydrochloride in 250 mlof dichloromethane was added 31.0 ml (22.2 mmol) of triethylamine. Themixture was cooled in an ice bath and trifluoroacetic anhydride (18.0ml, 127 mmol) was added over a period of 15-20 minutes. The mixture wasstirred at 0-10° C. for 1 hour and concentrated. The residue was dilutedwith 300 ml of ethyl acetate and saturated aqueous sodium bicarbonate(3×100 ml). The organic phase was dried over anhydrous magnesiumsulfate, filtered and concentrated to afford 26.5 g of the product as apale yellow oil (100%):

H-NMR (DMSO) 3.57 (3H, s), 3.37 (2H, t), 3.08 (1.9H, quartet, N--CH₃),2.93 (1.1H, s, N--CH₃), 2.30 (2H, t), 1.52 (4H, multiplet), and 1.23 ppm(2H, multiplet).

3. Preparation of methyl 6-N-methylamino-caproate hydrochloride. To asolution of 7.01 g (29.2 mmol) of N-6-(methylcaproyl)-trifluoroacetamidein 125 ml of anhydrous tetrahydrofuran was slowly added 1.75 g of 60%sodium hydride (43.8 mmol) in mineral oil. The mixture was stirred at15-25° C. for 30 minutes and then 6.2 g (43.7 mmol) of iodomethane wasadded. The mixture was stirred at 15-25° C. for 17 hours and thenfiltered through celite. The solids were rinsed with 50 ml oftetrahydrofuran. The filtrates were combined and concentrated. Theresidue was diluted with 150 ml of ethyl acetate and washed first with5% aqueous sodium sulfite (2×100 ml) and then with 100 ml of 1N aqueoushydrochloric acid. The organic phase was dried over anhydrous magnesiumsulfate, filtered and concentrated to afford a yellow oily residue. Theresidue was diluted with 250 ml of methanol and then hydrogen chloride(gas) was rapidly bubbled into the mixture for 2-3 minutes. Theresultant mixture was refluxed for 18 hours, cooled and concentrated.The residue was diluted with 150 ml of methanol and washed with hexane(3×150 ml) to remove mineral oil previously introduced with NaH. Themethanol phase was concentrated to afford 4.91 g of the product as ayellow oil (86%):

H-NMR (DMSO) 8.80 (2H, broad s), 3.58 (3H, s), 2.81 (2H, multiplet),2.48 (3H, s), 2.30 (2H, t), 1.52 (4H, multiplet), and 1.29 ppm (2H,multiplet).

4. Preparation of methyl 6-(N-methylcaproylamido-biotin.N-hydroxysuccinimidyl biotin (398 mg, 1.16 mmol) was added to a solutionof methyl 6-(N-methyl) aminocaproate hydrochloride (250 mg, 1.28 mmol)in DMF (4.0 ml) and triethylamine (0.18 ml, 1.28 mmol). The mixture washeated in an oil bath at 100° C. for 10 minutes. The solution wasevaporated, acidified with glacial acetic acid and evaporated again. Theresidue was chromatographed on a 25 mm flash chromatography columnmanufactured by Ace Glass packed with 50 g silica (EM Science,Gibbstown, N.J. particle size 0.40-0.63 mm) eluting with 15% MeOH/EtOAc.The product was obtained as a yellow oil (390 mg) in 79% yield.

5. Preparation of 6-(N-methyl-N-biotinyl) amino caproic acid. To asolution of methyl 6-(N-methylcaproylamido-biotin (391 mg, 1.10 mmol) inmethanol (2.5 ml) was added a 0.95N NaOH solution (1.5 ml). Thissolution was stirred at 23° C. for 3 hours. The solution was neutralizedby the addition of 1.0M HCl (1.6 ml) and evaporated. The residue wasdissolved in water, further acidified with 1.0M HCl (0.4 ml) andevaporated. The gummy solid residue was suspended in water and agitatedwith a spatula until it changed into a white powder. The powder wascollected by filtration with a yield of 340 mg.

6. Preparation of DOTA-benzylamido-N-methylcaproylamido-biotin. Asuspension of 6-(N-methyl-N-biotinyl)amino caproic acid (29 mg, 0.08mmol) and N-hydroxysuccinimide (10 mg, 0.09 mmol) in DMF (0.8 ml) washeated over a heat gun for the short time necessary for the solids todissolve. To this heated solution was added EDCI (15 mg, 0.08 mmol). Theresultant solution was stirred at 23° C. for 20 hours. To this stirredsolution were added aminobenzyl-DOTA (20 mg, 0.04 mmol) and pyridine(0.8 ml). The mixture was heated over a heat gun for 1 minute. Theproduct was isolated by preparative HPLC, yielding 3 mg.

E. Synthesis of a bis-DOTA conjugate with a preserved biotin carboxygroup.

A reaction scheme is shown below. ##STR45##

1. Preparation of methyl 6-bromocaproate (methyl 6-bromohexanoate).Hydrogen chloride (gas) was added to a solution of 5.01 g (25.7 mmol) of6-bromocaproic acid in 250 ml of methanol via vigorous bubbling for 2-3minutes. The mixture was stirred at 15-25° C. for 3 hours and thenconcentrated to afford 4.84 g of the product as a yellow oil (90%):

H-NMR (DMSO) 3.58 (3H, s), 3.51 (2H, t), 2.29 (2H, t), 1.78 (2H,pentet), and 1.62-1.27 ppm (4H, m).

2. Preparation of N,N-bis-(methyl 6-hexanoyl)-amine hydrochloride. To asolution of 4.01 g (16.7 mmol) of N-(methyl6-hexanoyl)-trifluoroacetamide (prepared in accordance with section D.2.herein) in 125 ml of anhydrous tetrahydrofuran was added 1.0 g (25 mmol)of 60% sodium hydride in mineral oil. The mixture was stirred at 15-25°C. for 1 hour and then 3.50 g (16.7 mmol) of methyl 6-bromocaproate wasadded and the mixture heated to reflux. The mixture was stirred atreflux for 22 hours. NMR assay of an aliquot indicated the reaction tobe incomplete. Consequently, an additional 1.00 g (4.8 mmol) of methyl6-bromocaproate was added and the mixture stirred at reflux for 26hours. NMR assay of an aliquot indicated the reaction to be incomplete.An additional 1.0 g of methyl 6-bromocaproate was added and the mixturestirred at reflux for 24 hours. NMR assay of an aliquot indicated thereaction to be near complete. The mixture was cooled and then directlyfiltered through celite. The solids were rinsed with 100 ml oftetrahydrofuran. The filtrates were combined and concentrated. Theresidue was diluted with 100 ml of methanol and washed with hexane(3×100 ml) to remove the mineral oil introduced with the sodium hydride.The methanol phase was treated with 6 ml of 10N aqueous sodium hydroxideand stirred at 15-25° C. for 3 hours. The mixture was concentrated. Theresidue was diluted with 100 ml of deionized water and acidified to pH 2with concentrated HCl. The mixture was washed with ether (3×100 ml). Theaqueous phase was concentrated, diluted with 200 ml of dry methanol andthen hydrogen chloride gas was bubbled through the mixture for 2-3minutes. The mixture was stirred at 15-25° C. for 3 hours and thenconcentrated. The residue was diluted with 50 ml of dry methanol andfiltered to remove inorganic salts. The filtrate was concentrated toafford 1.98 g of the product as a white solid (38%):

H-NMR (DMSO) 8.62 (2H, m) 3.58 (6H, s), 2.82 (4H, m) 2.30 (4H, t),1.67-1.45 (8H, m) and 1.38-1.22 ppm (4H, m).

3. Preparation of N,N'-bis-(methyl 6-hexanoyl)-biotinamide. To asolution of 500 mg (1.46 mmol) of N-hydroxysuccinimidyl biotin in 15 mlof dry dimethylformamide was added 600 mg (1.94 mmol) of N,N-bis-(methyl6-hexanoyl)amine hydrochloride followed by 1.0 ml of triethylamine. Themixture was stirred at 80-85° C. for 3 hours and then cooled andconcentrated. The residue was chromatographed on silica gel, elutingwith 20% methanol/ethyl acetate, to afford 620 mg of the product as anear colorless oil (85%):

H-NMR (CDCl₃) 5.71 (1H, s), 5.22 (1H, s), 4.52 (1H, m), 4.33 (1H, m),3.60 (3H, s), 3.58 (3H, s), 3.34-3.13 (5H, m), 2.92 (1H, dd), 2.75 (1H,d), 2.33 (6H, m) and 1.82-1.22 ppm (18H, m); TLC-R_(f) 0.39 (20:80methanol/ethyl acetate).

4. Preparation of N,N-bis-(6-hexanoyl)-biotinamide. To a solution of 610mg (0.819 mmol) of N,N-bis-(methyl 6-hexanoyl)-biotinamide in 35 ml ofmethanol was added 5.0 ml of 1N aqueous sodium hydroxide. The mixturewas stirred at 15-25° C. for 4.5 hours and then concentrated. Theresidue was diluted with 50 ml of deionized water acidified to pH 2 with1N aqueous hydrochloric acid at 4° C. The product, which precipitatedout as a white solid, was isolated by vacuum filtration and dried undervacuum to afford 482 mg (84%):

H-NMR (DMSO) 6.42 (1H, s), 6.33 (1H, s), 4.29 (1H, m), 4.12 (1H, m),3.29-3.04 (5H, m), 2.82 (1H, dd), 2.57 (1H, d), 2.21 (6H, m) and1.70-1.10 ppm (18H, m).

5. Preparation of N',N'-bis-(N-hydroxy-succinimidyl6-hexanoyl)-biotinamide. To a solution of 220 mg (0.467 mmol) ofN,N-bis-(6-hexanoyl)-biotinamide in 3 ml of dry dimethylformamide wasadded 160 mg (1.39 mmol) of N-hydroxysuccinimide followed by 210 mg(1.02 mmol) of dicyclohexyl-carbodiimide. The mixture was stirred at15-25° C. for 17 hours and then concentrated. The residue waschromatographed on silica gel, eluting with 0.1:20:80 aceticacid/methanol/ethyl acetate, to afford 148 mg of the product as a foamyoff-white solid (48%):

H-NMR (DMSO) 6.39 (1H, s), 6.32 (1H, s), 4,29 (1H, m), 4,12 (1H, m),3.30-3.03 (5H, m), 2.81 (9H, dd and s), 2.67 (4H, m), 2.57 (1H, d), 2.25(2H, t), 1.75-1.20 (18H, m); TLC-R_(f) 0.37 (0.1:20:80 aceticacid/methanol/ethyl acetate).

6. Preparation of N,N-bis-(6-hexanoylamidobenzyl-DOTA)-biotinamide. To amixture of 15 mg of DOTA-benzylamine and 6.0 mg ofN',N'-bis-(N-hydroxysuccinimidyl 6-hexanoyl)-biotinamide in 1.0 ml ofdry dimethylformamide was added 0.5 ml of dry pyridine. The mixture wasstirred at 45-50° C. for 4.5 hours and at 15-25° C. for 12 hours. Themixture was concentrated and the residue chromatographed on a 2.1×2.5 cmoctadecylsilyl (ODS) reverse-phase preparative HPLC column eluting witha--20 minute gradient profile of 0.1:95:5 to 0.1:40:60 trifluoroaceticacid:water:acetonitrile at 13 ml/minute to afford the desired product.The retention time was 15.97 minutes using the aforementioned gradientat a flow rate of 1.0 ml/minute on a 4.6 mm×25 cm ODS analytical HPLCcolumn.

F. Synthesis of an N-methyl-glycine linked conjugate.

A reaction scheme for this synthesis is shown below. ##STR46##

The N-methyl glycine-linked DOTA-biotin conjugate was prepared by ananalogous method to that used to prepare D-alanine-linked DOTA-biotinconjugates. N-methyl-glycine (trivial name sarcosine, available fromSigma Chemical Co.) was condensed with biotin-NHS ester in DMF andtriethylamine to obtain N-methyl glycyl-biotin. N-methyl-glycyl biotinwas then activated with EDCI and NHS. The resultant NHS ester was notisolated and was condensed in situ with DOTA-aniline and excesspyridine. The reaction solution was heated at 60° C. for 10 minutes andthen evaporated. The residue was purified by preparative HPLC to give[(N-methyl-N-biotinyl)-N-glycyl]-aminobenzyl-DOTA.

1. Preparation of (N-methyl)glycyl biotin. DMF (8.0 ml) andtriethylamine (0.61 ml, 4.35 mmol) were added to solids N-methyl glycine(182 mg, 2.05 mmol) and N-hydroxy-succinimidyl biotin (500 mg, 1.46mmol). The mixture was heated for 1 hour in an oil bath at 85° C. duringwhich time the solids dissolved producing a clear and colorlesssolution. The solvents were then evaporated. The yellow oil residue wasacidified with glacial acetic acid, evaporated and chromatographed on a27 mm column packed with 50 g silica, eluting with 30% MeOH/EtOAc 1%HOAc to give the product as a white solid (383 mg) in 66% yield.

H-NMR (DMSO): 1.18-1.25 (m, 6H, (CH₂)₃), 2.15, 2.35 (2 t's, 2H, CH₂ CO),2.75 (m, 2H, SCH₂), 2.80, 3.00 (2 s's, 3H, NCH₃), 3.05-3.15 (m, 1H,SCH), 3.95, 4.05 (2 s's, 2H, CH₂ N), 4.15, 4.32 (2 m's, 2H, 2CHN's),6.35 (s, NH), 6.45 (s, NH).

2. Preparation of [(N-methyl-N-biotinyl)glycyl]aminobenzyl-DOTA.N-hydroxysuccinimide (10 mg, 0.08 mmol) and EDCI (15 mg, 6.08 mmol) wereadded to a solution of (N-methylglycyl biotin (24 mg, 0.08 mmol) in DMF(1.0 ml). The solution was stirred at 23° C. for 64 hours. Pyridine (0.8ml) and aminobenzyl-DOTA (20mg, 0.04 mmol) were added. The mixture washeated in an oil bath at 63° C. for 10 minutes, then stirred at 23° C.for 4 hours. The solution was evaporated. The residue was purified bypreparative HPLC to give the product as an off white solid (8 mg, 0.01mmol) in 27% yield.

H-NMR (D₂ O): 1.30-1.80 (m, 6H), 2.40, 2.55 (2 t's, 2H, CH₂ CO),2.70-4.2 (complex multiplet), 4.35 (m, CHN), 4.55 (m, CHN), 7.30 (m, 2H,benzene hydrogens), 7.40 (m, 2H, benzene hydrogens).

G. Synthesis of a short chain amine-linked conjugate with a reducedbiotin carboxy group.

A two-part reaction scheme is shown below. ##STR47##

The biotin carboxyl group is reduced with diborane in THF to give aprimary alcohol. Tosylation of the alcohol with tosyl chloride inpyridine affords the primary tosylate. Aminobenzyl DOTA is acylated withtrifluoroacetic anhydride in pyridine to give(N-trifluoroacetyl)aminobenzyl-DOTA. Deprotonation with 5.0 equivalentsof sodium hydride followed by displacement of the biotin tosylateprovides the(N-trifluoracetamido-N-descarboxylbiotinyl)aminobenzyl-DOTA. Acidiccleavage of the N-trifluoroacetamide group with HCl(g) in methanolprovides the aminelinked DOTA-biotin conjugate.

EXAMPLE XVI Clearing Agent Evaluation Experimentation

The following experiments conducted on non-tumor-bearing mice wereconducted using female BALB/c mice (20-25 g). For tumor-bearing miceexperimentation, female nude mice were injected subcutaneously withLS-180 tumor cells, and, after 7 d, the mice displayed 50-100 mg tumorxenografts. The monoclonal antibody used in these experiments wasNR-LU-10. When radiolabeled, the NR-LU-10-streptavidin conjugate wasradiolabeled with I-125 using procedures described herein. Whenradiolabeled, PIP-biocytin was labeled with I-131 or I-125 usingprocedures described herein.

A. Utility of Asialoorosomucoid-Biotin (AO-Bt) in Reducing CirculatingRadioactivity from a Subsequently Administered Radiolabeled BiotinLigand. Mice bearing LS-180 colon tumor xenografts were injected with200 micrograms NR-LU-10 antibody-streptavidin (MAb-StrAv) conjugate attime 0, which was allowed to prelocalize to tumor for 22 hours. At thattime, 20 micrograms of AO-Bt was administered to one group of animals.Two hours later, 90 micrograms of a radioisotope-bearing,ligand-containing small molecule (PIP-biotin-dextran prepared asdiscussed in part B hereof) was administered to this group of mice andalso to a group which had not received AO-Bt. The results of thisexperiment with respect to radiolabel uptake in tumor and clearance fromthe blood indicated that tumor-targeting of the radiolabeledbiotin-containing conjugate was retained while blood clearance wasenhanced, leading to an overall improvement in amount delivered totarget/amount located in serum. The AUC tumor/AUC blood with clearingagent was 6.87, while AUC tumor/AUC blood without clearing agent was4.45. Blood clearance of the circulating MAb-StrAv conjugate wasenhanced with the use of clearing agent. The clearing agent wasradiolabeled in a separate group of animals and found to bind directlyto tumor at very low levels (1.7 pmol/g at a dose of 488 total pmoles(0.35%ID/g), indicating that it does not significantly compromise theability of tumor-bound MAb-StrAv to bind subsequently administeredradiolabeled ligand.

B. Preparation Protocol for PIP-Biotin-Dextran. A solution of 3.0 mgbiotin-dextran, lysine fixable (BDLF, available from Sigma Chemical Co.,St. Louis, Mo., 70,000 dalton molecular weight with approximately 18biotins/molecule) in 0.3 ml PBS and 0.15 ml 1M sodium carbonate, pH9.25, was added to a dried residue (1.87 mCi) of N-succinimidylp-I-125-iodobenzoate prepared in accordance with Wilbur, et al., J.Nucl. Med., 30: 216-226, 1989.

C. Dosing Optimization of AO-Bt. Tumored mice receiving StrAv-MAb asabove, were injected with increasing doses of AO-Bt (0 micrograms, 20micrograms, 50 micrograms, 100 micrograms and 200 micrograms). Tumoruptake of I-131-PIP-biocytin (5.7 micrograms, administered 2 hours afterAO-Bt administration) was examined. Increasing doses of AO-Bt had noeffect on tumor localization of MAb-StrAv. Data obtained 44 hours afterAO-Bt administration showed the same lack of effect. This data indicatesthat AO-Bt dose not cross-link and internalize MAb-StrAv on the tumorsurface, as had been noted for avidin administered followingbiotinylated antibody.

PIP-biocytin tumor localization was inhibited at higher doses of AO-Bt.This effect is most likely due to reprocessing and distribution to tumorof biotin used to derivatize AO-Bt. Optimal tumor to blood ratios (%injected dose of radiolabeled ligand/gram weight of tumor divided by %injected dose of radioligand/gram weight of blood were achieved at the50 microgram dose of AO-Bt. Biodistributions conducted followingcompletion of the protocols employing a 50 microgram AO-Bt dose revealedlow retention of radiolabel in all non-target tissues (1.2 pmol/g inblood; 3.5 pmol/gram in tail; 1.0 pmol/g in lung; 2.2 pmol/g in liver;1.0 pmol/g is spleen; 7.0 pmol/g in stomach; 2.7 pmol/g in kidney; and7.7 pmol/g in intestine). With 99.3 pmol/g in tumor, these resultsindicate effective decoupling of the PIP-biocytin biodistribution fromthat of the MAb-StrAv at all sites except tumor. This decouplingoccurred at all clearing agent doses in excess of 50 micrograms as well.Decreases in tumor localization of PIP-biocytin was the significantresult of administering clearing agent doses in excess of 50 micrograms.In addition, the amount of PIP-biocytin in non-target tissues 44 hoursafter administration was identical to localization resulting fromadministration of PIP-biocytin alone (except for tumor, where negligibleaccretion was seen when PIP-biocytin was administered alone), indicatingeffective decoupling.

D. Further Investigation of Optimal Clearing Agent Dose. Tumored miceinjected with MAb-StrAv at time 0 as above; 50 micrograms of AO-Bt attime 22 hours; and 545 microcuries of I-131-PIP-biocytin at time 25hours. Whole body radiation was measured and compared to that of animalsthat had not received clearing agent. 50 micrograms of AO-Bt wasefficient in allowing the injected radioactivity to clear from theanimals unimpeded by binding to circulating MAb-StrAv conjugate. Tumoruptake of I-131-PIP-biocytin was preserved at the 50 microgram clearingagent dose, with AUC tumor/AUC blood of 30:1 which is approximately15-fold better than the AUC tumor/AUC blood achieved in conventionalantibody-radioisotope therapy using this model.

E. Galactose- and Biotin-Derivatization of Human Serum Albumin (HSA).HSA was evaluated because it exhibits the advantages of being bothinexpensive and non-immunogenic. HSA was derivatized with varying levelsof biotin (1-about 9 biotins/molecule) via analogous chemistry to thatpreviously described with respect to AO. More specifically, to asolution of HSA available from Sigma Chemical Co. (5-10 mg/ml in PBS)was added 10% v/v 0.5M sodium borate buffer, pH 8.5, followed bydropwise addition of a DMSO solution of NHS-LC-biotin (Sigma ChemicalCo.) to the stirred solution at the desired molar offering (relativemolar equivalents of reactants). The final percent DMSO in the reactionmixture should not exceed 5%. After stirring for 1 hour at roomtemperature, the reaction was complete. A 90% incorporation efficiencyfor biotin on HSA was generally observed. As a result, if 3 molarequivalences of the NHS ester of LC-biotin was introduced, about 2.7biotins per HSA molecule were obtained. Unreacted biotin reagent wasremoved from the biotin-derivatized HSA using G-25 size exclusionchromatography. Alternatively, the crude material may be directlygalactosylated. The same chemistry is applicable for biotinylatingnon-previously biotinylated dextran.

HSA-biotin was then derivatized with from 12 to 15 galactoses/molecule.Galactose derivatization of the biotinylated HSA was performed accordingto the procedure of Lee, et al., Biochemistry, 15: 3956, 1976. Morespecifically, a 0.1M methanolic solution ofcyanomethyl-2,3,4,6-tetra-O-acetyl-1-thio-D-galactopyranoside wasprepared and reacted with a 10% v/v 0.1M NaOMe in methanol for 12 hoursto generate the reactive galactosyl thioimidate. The galactosylation ofbiotinylated HSA began by initial evaporation of the anhydrous methanolfrom a 300 fold molar excess of reactive thioimidate. Biotinylated HSAin PBS, buffered with 10% v/v 0.5M sodium borate, was added to the oilyresidue. After stirring at room temperature for 2 hours, the mixture wasstored at 4° C. for 12 hours. The galactosylated HSA-biotin was thenpurified by G-25 size exclusion chromatography or by buffer exchange toyield the desired product. The same chemistry is exploitable togalactosylating dextran. The incorporation efficiency of galactose onHSA is approximately 10%.

70 micrograms of Galactose-HSA-Biotin (G-HSA-B), with 12-15 galactoseresidues and 9 biotins, was administered to mice which had beenadministered 200 micrograms of StrAv-MAb or 200 microliters of PBS 24hours earlier. Results indicated that G-HSA-B is effective in removingStrAv-MAb from circulation. Also, the pharmacokinetics of G-HSA-B isunperturbed and rapid in the presence or absence of circulatingMAb-StrAv.

F. Non-Protein Clearing Agent. A commercially available form of dextran,molecular weight of 70,000 daltons, pre-derivatized with approximately18 biotins/molecule and having an equivalent number of free primaryamines was studied. The primary amine moieties were derivatized with agalactosylating reagent, substantially in accordance with the proceduretherefor described above in the discussion of HSA-based clearing agents,at a level of about 9 galactoses/molecule. The molar equivalenceoffering ratio of galactose to HSA was about 300:1, with about one-thirdof the galactose being converted to active form. 40 Micrograms ofgalactose-dextran-biotin (GAL-DEX-BT) was then injected i.v. into onegroup of mice which had received 200 micrograms MAb-StrAv conjugateintravenously 24 hours earlier, while 80 micrograms of GAL-DEX-BT wasinjected into other such mice. GALDEX-BT was rapid and efficient atclearing StrAv-MAb conjugate, removing over 66% of circulating conjugatein less than 4 hours after clearing agent administration. An equivalenteffect was seen at both clearing agent doses, which correspond to 1.6(40 micrograms) and 3.2 (80 micrograms) times the stoichiometric amountof circulating StrAv conjugate present.

G. Dose Ranging for G-HSA-B Clearing Agent. Dose ranging studiesfollowed the following basic format:

200 micrograms MAb-StrAv conjugate administered;

24 hours later, clearing agent administered; and

2 hours later, 5.7 micrograms PIP-biocytin administered.

Dose ranging studies were performed with the G-HSA-B clearing agent,starting with a loading of 9 biotins per molecule and 12-15 galactoseresidues per molecule. Doses of 20, 40, 70 and 120 micrograms wereadministered 24 hours after a 200 microgram dose of MAb-StrAv conjugate.The clearing agent administrations were followed 2 hours later byadministration of 5.7 micrograms of I-131-PIP-biocytin. Tumor uptake andblood retention of PIP-biocytin was examined 44 hours afteradministration thereof (46 hours after clearing agent administration).The results showed that a nadir in blood retention of PIP-biocytin wasachieved by all doses greater than or equal to 40 micrograms of G-HSA-B.A clear, dose-dependent decrease in tumor binding of PIP-biocytin ateach increasing dose of G-HSA-B was present, however. Since nodose-dependent effect on the localization of MAb-StrAv conjugate at thetumor was observed, this data was interpreted as being indicative ofrelatively higher blocking of tumor-associated MAb-StrAv conjugate bythe release of biotin from catabolized clearing agent. Similar resultsto those described earlier for the asialoorosomucoid clearing agentregarding plots of tumor/blood ratio were found with respect to G-HSA-B,in that an optimal balance between blood clearance and tumor retentionoccurred around the 40 microgram dose.

Because of the relatively large molar amounts of biotin that could bereleased by this clearing agent at higher doses, studies were undertakento evaluate the effect of lower levels of biotinylation on theeffectiveness of the clearing agent. G-HSA-B, derivatized with either 9,5 or 2 biotins/molecule, was able to clear MAb-StrAv conjugate fromblood at equal protein doses of clearing agent. All levels ofbiotinylation yielded effective, rapid clearance of MAb-StrAv fromblood.

Comparison of these 9-, 5-, and 2-biotin-derivatized clearing agentswith a single biotin G-HSA-B clearing agent was carried out in tumoredmice, employing a 60 microgram dose of each clearing agent. Thisexperiment showed each clearing agent to be substantially equallyeffective in blood clearance and tumor retention of MAb-StrAv conjugate2 hours after clearing agent administration. The G-HSA-B with a singlebiotin was examined for the ability to reduce binding of a subsequentlyadministered biotinylated small molecule (PIP-biocytin) in blood, whilepreserving tumor binding of PIP-biocytin to prelocalized MAb-StrAvconjugate. Measured at 44 hours following PIP-biocytin administration,tumor localization of both the MAb-StrAv conjugate and PIP-biocytin waswell preserved over a broad dose range of G-HSA-B with onebiotin/molecule (90 to 180 micrograms). A progressive decrease in bloodretention of PIP-biocytin was achieved by increasing doses of the singlebiotin G-HSA-B clearing agent, while tumor localization remainedessentially constant, indicating that this clearing agent, with a lowerlevel of biotinylation, is preferred. This preference arises because thesingle biotin G-HSA-B clearing agent is both effective at clearingMAb-StrAv over a broader range of doses (potentially eliminating theneed for patient-to-patient titration of optimal dose) and appears torelease less competing biotin into the systemic circulation than thesame agent having a higher biotin loading level.

Another way in which to decrease the effect of clearing agent-releasedbiotin on active agent-biotin conjugate binding to prelocalizedtargeting moiety-streptavidin conjugate is to attach the protein orpolymer or other primary clearing agent component to biotin using aretention linker. A retention linker has a chemical structure that isresistant to agents that cleave peptide bonds and, optionally, becomesprotonated when localized to a catabolizing space, such as a lysosome.Preferred retention linkers of the present invention are short stringsof D-amino acids or small molecules having both of the characteristicsset forth above. An exemplary retention linker of the present inventionis cyanuric chloride, which may be interposed between an epsilon aminogroup of a lysine of a proteinaceous primary clearing agent componentand an amine moiety of a reduced and chemically altered biotin carboxymoiety (which has been discussed above) to form a compound of thestructure set forth below. ##STR48## When the compound shown above iscatabolized in a catabolizing space, the heterocyclic ring becomesprotonated. The ring protonation prevents the catabolite from exitingthe lysosome. In this manner, biotin catabolites containing theheterocyclic ring are restricted to the site(s) of catabolism and,therefore, do not compete with active-agent-biotin conjugate forprelocalized targeting moiety-streptavidin target sites.

Comparisons of tumor/blood localization of radiolabeled PIP-biocytinobserved in the G-HSA-B dose ranging studies showed that optimal tumorto background targeting was achieved over a broad dose range (90 to 180micrograms), with the results providing the expectation that even largerclearing agent doses would also be effective. Another key result of thedose ranging experimentation is that G-HSA-B with an average of only 1biotin per molecule is presumably only clearing the MAb-StrAv conjugatevia the Ashwell receptor mechanism only, because too few biotins arepresent to cause cross-linking and aggregation of MAb-StrAv conjugatesand clearing agents with such aggregates being cleared by thereticuloendothelial system.

H. Tumor Targeting Evaluation Using G-HSA-B. The protocol for thisexperiment was as follows:

Time 0: administer 400 micrograms MAb-StrAv conjugate;

Time 24 hours: administer 240 micrograms of G-HSA-B with one biotin and12-15 galactoses and

Time 26 hours: administer 6 micrograms of ##STR49## Lu-177 is complexedwith the DOTA chelate using known techniques therefor.

Efficient delivery of the Lu-177-DOTA-biotin small molecule wasobserved, 20-25% injected dose/gram of tumor. These values areequivalent with the efficiency of the delivery of the MAb-StrAvconjugate. The AUC tumor/AUC blood obtained for this non-optimizedclearing agent dose was 300% greater than that achievable by comparabledirect MAb-radiolabel administration. In addition, the HSA-basedclearing agent is expected to exhibit a low degree of immunogenicity inhumans.

EXAMPLE XVII Synthesis of DOTA Chelates

A. Peptide cyclization.

N-Boc-triglycine. To a suspension of 18.92 g (100 mmole) of triglycine(available from Sigma Chemical Company) in 100 ml dioxane and 100 mlwater was added 27.88 ml (200 mmole, 2.0 equivalents) of triethylamineand 27.1 g (110 mmole, 1.1 equivalents) of2-[tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (available fromFluka, N.Y.). The reaction mixture was stirred at room temperatureovernight. Water (65 ml) was added to the mixture, and the resultantmixture was extracted with ether (4×65 ml) ant EtoAc (1×70 ml). Theaqueous layer was adjusted to pH 1.5 with 6M HCl at 0° C. and thenextracted with EtOAc (25×100 ml) by the salt-out technique. Asubstantial amount of product precipitated out of the EtOAc extractionand was collected by filtration. The EtOAc filtrate was reduced to 1/5of its original volume under vacuum to produce additional precipitate.The precipitate was collected by filtration and the above procedure(reduction of volume to 1/5) was repeated. The precipitates werecombined and dried under vacuum to give 23.82 g of product. (82.3%yield) ¹ H-NMR (CD₃ CN, δ): 7.05 (bs, 2H, NH), 5.70 (bs, 1H, NH), 3.82(dd, 4H, CH₂), 3.64 (d, 2H, CH₂) and 1.92 (s, 9H, t-butyl).

N-Boc-triglycine N-hydroxysuccinimide ester. To a turbid solution of17.357 g (60 mmole) of N-BOC-triglycine and 13.5 g (65.4 mmole, 1.09equivalents) of 1,3-dicyclohexylcarbodiimide (DCC) in 300 ml anhydrousdioxane was added 6.906 g (60 mmole, 1 equivalent) ofN-hydroxysuccinimide in one portion. The reaction mixture was stirred atroom temperature overnight under nitrogen. DCU, dicyclohexylurea, wasremoved by filtration, and the filtrate was concentrated to drynessunder vacuum. The resulting foamy product was triturated with hexane andthen further dried under vacuum to give 27.68 g of crude product. (>100%crude yield) ¹ H-NMR studies of this crude product showed that it mightbe formed in a 2:1 complex with dioxane. ¹ H-NMR (CDCl₃,δ): 7.34 and7.06 (2m, NH), 5.40 (t, NH), 4.36 (d, CH₂), 4.04 (d, CH₂) 3.82 (d, CH₂),3.71 (s, dioxane), 2.85 (s, CH₂), and 1.70 (s, t-butyl).

N-Boc-triglycine-p-nitro-phenylalanine. To a stirred solution of 30mmole of N-boc-triglycine N-hydroxysuccinimide ester and 8.828 g (42mmole, 1.4 equivalents) of p-nitrophenylalanine (available from AldrichChemical Co., Milwaukee) in 56 ml of DMF and 114 ml water was added 5.04g (60 mmole, 2.0 equivalents) of NaHCO₃. The reaction mixture wasstirred at room temperature for 5 hours. Precipitate was filtered off,and the filtrate was concentrated under vacuum. The resulting residuewas dissolved in 250 ml of water and acidified with 6M HCl at 0° C. topH 1.4. The aqueous layer was extracted with EtOAc (6×150 ml). Theorganic layers were combined and evaporated to dryness under vacuum. Thecrude product thus obtained was crystallized in 2-propanol to form theproduct. ¹ H-NMR (CD₃ CN,δ) 8.12 and 7.48 (2d, 4H, ArH), 7.20 (m, 3H,NH), 5.78 (m, 1H, NH), 4.64 (m, 1H, CH), 3.72 (m, 6H, CH₂), 3.20 (m, 2H,CH₂ Ar), and 1.93 (s, 9H, t-butyl).

The Boc protecting group of the linear tetrapeptideN-Boc-triglycine-p-nitro-phenylalanine is cleaved using 50%trifluoroacetic acid in CH₂ Cl₂ at room temperature for 30 minutes toform triglycine-pnitrophenyl-alanine. Peptide cyclization is thenachieved by mixing triglycine-p-nitrophenyl-alanine with 1.2 equivalentsof diphenylphosphoroazide (DPPA) and 10 equivalents of sodiumbicarbonate in anhydrous DMF under high dilution conditions (0.007M oftriglycine-p-nitrophenyl-alanine) at 4° C. for three days. Water andBio-RAd AG501-X8 mixed bed ion exchange resin are added to the reactionmixture, and the mixture is stirred at room temperature for 3 hours. Theresin is separated by filtration, and the filtrate is concentrated invacuo to give a crude product. The crude product is extracted with HOActwice, and the HOAc extractions are combined and dried under vacuum togive the cyclo-tetrapeptide2-(4-nitrobenzyl)-3,6,9,12-tetraoxo-1,4,7,10-tetraazacyclododecane.

As a consequence of the relatively poor solubility of thenitro-derivatized compound2-(4-nitrobenzyl-3,6,9,12-tetraoxo-1,4,7,10-tetraazacyclododecane,alternative intermediates may be employed in the synthesis ofnitrobenzyl-DOTA. For example, the nitro group may be substituted byhydrogen or RNH- where R is an amino protecting group. Preferred Rgroups include toluenesulfonyl, isonicotinyl-carbamate and9-fluorenylmethyl carbamate. These alternative compounds can be preparedanalogously to the nitro derivative by substituting p-nitrophenylalaninewith phenylalanine (available from Sigma Chemical Co., St. Louis Mo. forhydrogen substitution) or the protected p-aminophenylalanine (for RNH-substitution and prepared, for example, as follows: p-nitrophenylalanineis reacted with BOC-ON in dioxane and water in the presence of Et₃ N atroom temperature overnight to afford N-Boc-p-nitrophenylalanine. Thenitro group of N-Boc-p-nitro-phenylalanine is reduced with 10% Pd-Cunder 40 psi of H₂ in MeOH for 2 hours to produce theα-N-Boc-p-amino-phenylalanine. Subsequent protection of the para-aminogroup is achieved by treating α-N-Boc-p-amino-phenylalanine with4-methoxy-2,3,6-trimethylbenzene-sulfonylchloride (MtrCl) and Et₃ N inCH₂ Cl₂ at room temperature for 17 hours. The Boc protecting group ofthe alpha-amino group of α-N-Boc-p-Mtr-amidophenyl-alanine is thencleaved by 50% TFA in CH₂ Cl₂ at room temperature for 30 minutes to givethe Mtr protected p-aminophenylalanine).

When hydrogen substitution is employed, the nitro group is introducedlater in the synthesis (e.g., following cyclization and reduction toform the tetraamine product). When the protected amino intermediate isemployed, the amine protecting group is inert to diborane reduction.Consequently, alkylation with bromoacetic acid followed by deprotectionof the para-amino protecting group afford an anilino-DOTA product.

The tetraamine 2-(p-nitrophenyl)-1,4,7,10-tetraaza-cyclododecane isformed by reducing2-(4-nitrobenzyl)-3,6,9,12-tetraoxo-1,4,7,10-tetraazacyclododecane with12 equivalents of diborane in THF at 50° C. for two days. Hydrolysis ofthe resulting borane complex is accomplished in MeOH saturated with HClgas. 2-(p-nitrophenyl)-1,4,7,10-tetraazacyclododecane is treated with4.4 equivalents of bromoacetic acid in water at pH 10 at 70° C. for oneday to give crude product. The product p-nitrobenzyl-DOTA is obtainedafter anion-exchange column chromatography with elution of increasingconcentration of ammonium acetate, for example.

B. Diethylenetriamine and N-alkylated phenyl alanine route.

N, N', N'"-tris-(p-toluenesulfonyl)-diethylene triamine. A solution of1N sodium hydroxide is added to a solution of diethylene triamine(available from Aldrich Chemical Co., Milwaukee, Wis.) in water untilthe pH is 10. Then p-toluenesulfonyl chloride (1.4 mole equivalents) isadded as a solid all at once. Additional 1N sodium hydroxide is added asneeded to maintain the pH of the reaction mixture between 10 and 12.When the pH ceases to change the mixture is filtered. The solid isrinsed with dichloromethane. The resultant solid is dried in vacuo togive the product which, if necessary, may be purified byrecrystallization.

N-(p-toluenesulfonyl)-(4-nitrophenyl)-alanine. To a solution of4-nitro-L-phenyl-alanine (available from Aldrich) (100g) in water (3.0ml) and 0.95N NaOH (5.0 ml) was added p-toluenesulfonyl chloride (1.27g) all at once. The reaction was stirred at room temperature, and the pHwas continuously monitored with a pH meter. After 2 hours, the pH of thereaction had dropped from 10.5 to 8.3; additional 0.95N NaOH was addedto raise the pH to 10.4. After 2 more hours, the pH was 8.5, andadditional 0.95N NaOH was added to raise the pH to 11.0. The reactionmixture was stirred an additional 16 hours at room temperature. Thereaction mixture which contained a large amount of white solid, wasdiluted with water (30 ml) and CH₂ Cl₂ (100 ml) and filtered. The solidwas rinsed with CH₂ Cl₂. The filtrate was transferred to a separatoryfunnel. The CH₂ Cl₂ layer, which contained p-toluenesulfonyl chloride,was separated and discarded. The aqueous and filtered solid phases werecombined, acidified with 1.0M HCl to pH 2, and filtered. The solid wasdried in vacuo to give 1.55 g product (98% yield). ¹ H-NMR (CDCl₃): 2.40(s, 3H, CH₃), 3.18 (dd, 2H, CH₂), 4.25 (m, 1H, CH), 5.25 (d, 1H, NH),7.25 (dd, 4H), 7.60 (d, 2H), and 8.05 (d, 2H).

N-(p-toluenesulfonyl)-N-(2-hydroxyethyl)-(4-nitrophenyl)-L-alanine. DMFwas added to a mixture of N-tosyl-p-nitrophenylalanine (332 mg, 1.0mmol), ethylene carbonate (440 mg, 5.0 mmol), and potassium carbonate(690 mg, 5.0 mmol) and the resultant mixture was heated in an oil bathat 60° C. for 19 hours. The DMF was evaporated under reduced pressure.The residue was diluted with 1.0M HCl (30 ml) and extracted withmethylene chloride (3×30 ml). The combined CH₂ Cl₂ extracts were dried(MgSO₄) and evaporated to give 430 mg brown oil which was purified byflash chromatography on silica gel with 99:1 ethyl acetate:acetic acidto give the product in 40% yield. ¹ H NMR (CDCl₃): 2.40 (s, 3H, CH₃),3.20 (dd, 2H, ArCH₃), 4.32 (m, 1H, CHN), 4.50-4.85 (m, 3H), 5.70 (m,1H), 7.17 (d, 2H), 7.30 (d, 2H), 7.55 (d, 2H), 8.03 (d, 2H). IR (CDCl₃):3300, 1755, 1670, 1420, 1160 cm⁻¹.

1-(4-Nitrobenzyl)-N-tosyl-iminodiethanol. 1.0M Diborane in THF is addedto a solution ofN-(p-toluenesulfonyl)-N-(2-hydroxyethyl)-(4-nitrophenyl)-L-alanine inTHF. The reaction is stirred at 23° C. for 4 hours. Methanol is addedand the solution is evaporated. The residue is treated with methanol andevaporated twice more to dryness. The product is purified bychromatography on silica gel (Silica gel 60, EM Science, Gibbstown,N.J.).

N-[2-(p-tolylsulfonyl)oxy]ethyl-N-[2-p-tolylsulfonyl)oxy]-1-(4-nitrobenzyl)ethyl]-p-toluenesulfonamide.P-toluenesulfonyl chloride is added to a solution of1-(4-nitrobenzyl)-N-(p-toluenesulfonyl)-iminodiethanol indichloromethane and triethylamine. The solution is stirred at 23° C. for20 hours, diluted with methylene chloride and washed twice with 0.1MHCl. The methylene chloride extracts are dried (MgSO₄) and evaporated.The residue is purified by chromatography on silica gel (Silica gel 60,EM Science).

(S)-2-(p-nitrobenzyl)-N,N',N",N'"-tetrakis-(tolylsulfonyl)-1,4,7,10-tetraazacyclododecane.Equimolar amounts ofN-[2-(p-tolylsulfonyl)oxy]ethyl-N-[2-(p-tolylsulfonyl)oxy]-1-(4-nitrobenzyl)ethyl]-p-toluenesulfonamideand N,N',N"N'"-tris-(p-toluene sulfonyl)-diethylenetriamine aredissolved in DMF. Two equivalents of cesium carbonate are added and thereaction is heated at 75° C. for 14 hours. The solvent is removed underreduced pressure, and the residue is dissolved in CHCl₃ and washed with0.1M HCl three times. The CHCl₃ phase is dried (MgSO₄) and evaporated.The residue is purified on silica gel (Silica gel 60, EM Science) withCHCl₃. Conversion of this product to nitro-benzyl-DOTA is accomplishedvia the procedure discussed in Renn et al., Bioconj. Chem., 3:563-569,1992 or as discussed in subpart C set forth below.

In prior art synthetic routes, N-tosylation of an aminoalcohol proved tobe problematic and proceeded in low yield. The synthetic route set forthabove, in contrast, features selective N-tosylation prior tointroduction of the alcohol. The subsequent O-tosylation step is lesssusceptible to potential side reactions arising from nucleophilic attackof the amino group on the O-tosylate. Also, this synthetic route has theadvantage that the electron withdrawing nitrobenzyl group is substitutedon the electrophile (i.e., the leaving group half of the moleculeundergoing cyclization), thus not impairing the nucleophilicity of thenucleophilic tosylamide half. When this substitution is reversed, theyield of the cyclization step is greatly reduced. This synthetic routeincludes seven steps and is, therefore, relatively efficient.

C. Tosylation cyclization.

N-Boc-triglycine-p-nitro-phenylalanine is prepared in accordance withthe procedure described in subpart A of this Example.N-Boc-triglycine-p-nitro-phenylalanine is first treated with HClgas-saturated dioxane to cleave the Boc protecting group, forming adeprotected triamide intermediate. This intermediate is then reducedwith 13 equivalents of diborane in THF at refluxing temperature.Hydrolysis of the resultant borane complex with MeOH saturated with HClgas affords a hydroxy tetramine compound,2-(p-nitrobenzyl)-3,6,9,12-tetraazadodecanol.

Tosylation of 2-(p-nitrobenzyl)-3,6,9,12-tetraazadodecanol is carriedout in a stepwise fashion as set forth below. The hydroxy group is firsttransiently protected with a trimethylsilyl group by treating2-(p-nitrobenzyl)-3,6,9,12-tetraazadodecanol with 1.2 equivalents oftrimethylchlorosilane (available from Aldrich, Milwaukee, Wis.) and 2equivalents of triethylamine in THF at room temperature for 4 hours.Solvent is removed from the resultant mixture under reduced pressure(e.g., 1 torr). The transient protected intermediate is reacted with 4equivalents of p-toluenesulfonyl chloride and 4 equivalents oftriethylamine in CH₂ Cl₂ /CH₃ CN (2/1 v/v) at room temperature for 5hours to afford an N-tosylated intermediate. The transienttrimethylsilyl protecting group is then cleaved, and the free hydroxyderivative is converted to the O-tosylated, N-tosylated compound IX,2-(p-nitrobenzyl)-N,N',N",N'",O-pentakis(tolyl-sulfonyl)-3,6,9,12-tetraazadodecanol,by reacting the N-tosylated intermediate with 1.1 equivalents ofp-toluenesulfonic chloride and 1 equivalent of triethylamine in CH₂ Cl₂.The transient protection of the hydroxy group is conducted to increasethe yield of2-(p-nitrobenzyl)-N,N',N",N'",O-pentakis(tolyl-sulfonyl)-3,6,9,12-tetraazadodecanolby minimizing the undesired side reactions potentially arising frominter- and intra-molecular condensations of partially tosylatedintermediates.

The intramolecular cyclization step undertaken to give2-(p-nitrobenzyl)-N,N',N",N'"-tetrakis(tolylsulfonyl)-1,4,7,10-tetraazacyclododecane is performed by treating2-(p-nitrobenzyl)-N,N',N",N'",O-pentakis(tolylsulfonyl)-3,6,9,12-tetraazadodecanol with 1 equivalentof CsCO₃ in DMF under high dilution conditions (0.01M of2-(p-nitrobenzyl)-N,N',N",N'",O-pentakis(tolylsulfonyl)-3,6,9,12tetraazadodecanol) at 60° C. for 5hours under nitrogen. The tosyl protecting groups of2-(p-nitrobenzyl)-N,N',N",N'"-tetrakis(tolylsulfonyl)-1,4,7,10-tetraazacyclododecane are removed usingconcentrated H₂ SO₄ at 100° C. for 56 hours, affording2-(p-nitrophenyl)-1,4,7,10-tetraazacyclododecane2-(p-nitrophenyl)-1,4,7,10-tetraazacyclododecane is treated with 4.4equivalents of bromoacetic acid in water at pH 10 at 70° C. for one dayto give crude product. The product p-nitrobenzyl-DOTA is obtained afteranion-exchange column chromatography with elution of increasingconcentration of ammonium acetate, for example.

D. Intramolecular cyclization of nitro-phenylalanine-(glycine)₃.

(S)-p-nitrophenylalanylglycylglycylglycine is prepared in accordancewith the method described in Renn and Meares, Bioconj. Chem., 3:563-569, 1992.

2-(4-Nitrobenzyl)-3,6,9,12-tetraoxo-1,4,7,10-tetraazacyclododecane.Diphenylphosphorylazide (available from Aldrich, Milwaukee, Wis.) isadded to a stirred solution of(S)-p-nitrophenyl-alanylglycyl-glycylglycine in DMF at 0° C.Triethylamine is then added to the reaction. Stirring at 0° C. iscontinued for 4 hours and is followed by stirring at room temperaturefor 14 hours. The solvent is removed under reduced pressure. The residueis taken up in glacial acetic acid. The slurry is heated to 45° C. andfiltered. The solid product is collected by filtration.

(S)-2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane. Borane-pyridine(available from Aldrich) is added to a suspension of2-(4-nitrobenzyl)-3,6,9,12-tetraoxo-1,4,7,10-tetraazacyclododecane inpyridine. The mixture is heated under reflux at 120° C. for 14 hours.The solvent is removed under reduced pressure. The residue is taken upin methanol and heated under reflux for 2 hours. The solvent is removedunder reduced pressure. The residue is dissolved in methanol andre-evaporated. The solid residue is purified by chromatography onreverse phase Baker G18 silica gel available from J.T. Baker, Inc.,Phillipsburg, N.J. Conversion of this product to nitro-benzyl-DOTA isaccomplished via the procedure discussed in Renn et al., Bioconj. Chem.,3:563-569, 1992 or as set forth is subpart A above.

This synthetic route includes five steps and is, therefore, quiteefficient. In prior art synthetic routes, the yield-limiting step is anintermolecular cyclization, while the route described above involvesintra-molecular cyclization. Generally, intra-molecular cyclizationsproceed in higher yield than inter-molecular cyclizations. In addition,intra-molecular cyclizations do not require the use of high dilutiontechniques.

E. Phenyl-alanine route.

As an alternative to employing 4-nitrophenylalanine in synthetic routesto benzyl-substituted tetraazadodecane derivatives, phenylalanine may beused. Two advantages may be gained by introducing the nitro groupfollowing cyclization. First, the solubility of the syntheticintermediates in organic solvents may be improved. Second, cyclizationstep yield may be enhanced. See, for example, McMurry et al., Bioconj.Chem., 3: 108-117, 1992.

An exemplary synthesis of this type is set forth below, whereinphenylalanine (available from Aldrich) was substituted fornitrophenylalanine in Route A set forth above. Briefly,N-Boc-triglycyl-N-hydroxysuccinimidyl ester obtained as set forth insubpart A of this Example is condensed with phenylalanine to giveN-Boc-triglycylphenylphenylalanine. The Boc protecting group is cleavedwith trifluoroacetic acid to give N-Boc-triglycinephenylalanine.Intramolecular cyclization of 3 affords the benzyl-cyclododecanederivative 2-(benzyl)-3,6,9,12-tetraoxo-1,4,7,10-tetraazacyclododecane,which is nitrated with nitric acid in sulfuric acid to give2-(4-nitrobenzyl)-3,6,9,12-tetraoxo-1,4,7,10-tetraazacyclododecane.Alternatively, a milder nitration reagent, such as NO₂ ⁺ CF₃ SO₃ ⁻, maybe employed. The amide carbonyls are reduced with borane-pyridine atelevated temperature. Conversion of the tetraazacyclododecane tonitrobenzyl-DOTA is accomplished via the procedure discussed in Renn etal., Bioconj. Chem., 3:563-569, 1992 or as discussed in subsection A setforth above.

F. Phenyl-lactic acid route.

Phenyl-lactic acid (available from Aldrich Chemical Co., Milwaukee,Wis.) is nitrated with nitric acid in sulfuric acid. P-nitrophenyllactic acid is esterified with methanol and gaseous hydrochloric acid.Methyl-p-nitrophenyl lactate is condensed with ethylene diamine toprovide the amide adduct,3-(4-nitrophenyl)-2-hydroxy-N-(2-aminoethyl)propionamide.

The free amino group of the amide adduct is acylated with N-hydroxysuccinimidyl-N-trifluoroacetyl glycylglycine, formed through thederivatization of diglycine (available from Sigma Chemical Co., St.Louis, Mo.) as described below. The amino group of diglycine isprotected with a BOC protecting group using BOC-ON (available fromAldrich Chemical Co., Milwaukee, Wis.) in aqueous DMF. The Bocdiglycineadduct is then converted to the N-hydroxysuccinimidyl ester employingdicyclohexylcarbodiimide and N-hydroxysuccinimide.

The aforementioned free amino acylation affords3-(4-nitrophenyl)-2-hydroxy-N-(trifluoroacetylglycylglycyl-2-aminoethyl)propionamide.The hydroxyl group of3-(4-nitrophenyl)-2-hydroxy-N-(trifluoroacetylglycylglycyl-2-aminoethyl)propionamideis tosylated with TsCl in pyridine to give3-(4-nitrophenyl)-2-(p-toluenesulfonyl)-oxy-N-(trifluoroacetylglycylglycyl-2-aminoethyl)-propionamide.The trifluoroacetylamide group of3-(5-nitrophenyl)-2-(p-toluenesulfonyl)-oxy-N-(trifluoroacetylglycylglycyl-2-aminoethyl)-propionamideis deprotonated with sodium hydride and cyclized to give2-(4-nitrobenzyl)-3,8,11-trioxo-N-trifluoroacetyl-1,4,7,10-tetraazadodecane.The trifluoroacetyl group of2-(4-nitrobenzyl)-3,8,11-trioxo-N-trifluoroacetyl-1,4,7,10-tetraazacyclododecaneis cleaved with sodium hydroxide to afford a triamide product,2-(4-nitrobenzyl)-3,8,11-trioxo-1,4,7,10-tetraazacyclododecane. Thetriamide is reduced with borane to give2-(p-nitrophenyl)-1,4,7,10-tetraazacyclododecane. Conversion of thisproduct to nitro-benzyl-DOTA is accomplished via the procedure discussedin the Renn et al. article referenced above or as discussed insubsections A and C set forth above.

This synthetic route employs a phenyl-lactic acid starting material andpeptide synthesis steps that are synthetically facile. Theintramolecular cyclization is not conducted under high dilutionconditions and a cyclomonomer product is favored.

EXAMPLE XVIII Y-90 Chelation and Radiolabeled Biotinylated Molecules

A. Radiolysis Experimentation.

The stability of Y-90-DOTA-biotin conjugate to radiolysis was examinedat different levels of specific activity (from 10 mCi/mg to 875 mCi/mg).Specific activity is the ratio of yttrium activity (mCi) to the mass ofthe DOTA-biotin conjugate (mg). The effect of radioprotectants, such asgentisic acid and ascorbic acid was also examined at the same range ofspecific activity.

The results summarized in Tables 5 and 6 were obtained by the proceduredescribed below. To the desired starting Y-90 activity, an appropriateamount of DOTA-biotin conjugate, dissolved in 0.5M, pH 5 ammoniumacetate, was added. If employed, a radioprotectant was either added tothis mixture or added to the radiolabeled preparation following theradiolabeling procedure. The composition of each reaction mixture (e.g.,activity, chelate linker, total volume, radioprotectant and the like)were as set forth in the Tables. A 2 ml centrifuge tube was used as areaction vessel, and the reaction mixture was incubated at 80° C. in awater bath for 30 minutes. Radiopurities were assessed by HPLC atdifferent time intervals.

High-performance liquid chromatography (HPLC) was conducted with aBeckman Model 110B solvent delivery system and Beckman Model 170radioisotpe detector. Reverse-phase HPLC chromatography was carried outusing a Beckman ultrasphere (4.6 mm×12.5 cm) C-18 column using agradient solvent system at a flow rate of 1.0 ml/min. Solvent A in thegradient was a 10 mM diethylenetriaminepenta-acetic acid calciumtrisodium salt water solution. Solvent B was 60% acetonitrile in a 10 mMdiethylenetriaminepenta-acetic acid calcium trisodium salt watersolution. The gradient was increased to 75% B over 15 minutes andmaintained at 75% B for an additional 15 minutes. The gradient wasdecreased to the original 100% A over a 10 minute time period.

Table 5 indicates results of experimentation when the radioprotectant isadded following radiolabeling, while Table 6 shows experimental resultswhen the radioprotectant is added as a radiolabeling ingredient. Tables5 and 6 demonstrate the instability of Y-90-DOTA-biotin conjugate (withN-Me ligand indicating a conjugate wherein biotin is bound to DOTA viaan N-methyl-glycine linker as set forth in Example XV(F) above and LCligand indicating a conjugate wherein biotin is bound to DOTA via--NH--(CH₂)₅, long chain DOTA-biotin) to radiolysis as well as thestability of such conjugates provided by the radioprotectants ascorbicacid and gentisic acid. Low specific activity preparations did notexhibit problematic radiolysis. High specific activity preparations;however, exhibited radiolysis. Addition of either gentisic acid orascorbic acid decreased radiolysis in high specific activitypreparations, with ascorbic acid giving somewhat better results thangentisic acid.

                                      TABLE 5                                     __________________________________________________________________________                          Specific                                                     Activity Volume Activity                                                   Antioxidant Isotope Ligand mCi uL mCi/mg T = 0 T = 1 T = 4 T = 24 T =       __________________________________________________________________________                                           48                                     No    In  N--Me                                                                             0.400                                                                             165 10  91 -- -- 90  --                                       No In N--Me 2.2 300 100 94 -- -- 92 --                                        No Y N--Me 0.15 85 10 98 -- 98 96 94                                          No Y N--Me 1.5 79 100 97 -- 94 76 61                                          No Y N--Me 5.0 200 135 98 -- -- 70 --                                         A.A. Y N--Me 5.0 200 135 98 -- -- 98 --                                       No Y N--Me 3.7 40 875 89 85 73 -- --                                          A.A. Y N--Me 3.5 40 875 89 -- 89 89 --                                        No Y L.C. 3.5 40 875 79 73 59 -- --                                           A.A. Y L.C. 3.5 40 875 79 -- 79 79 --                                       __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________                            Specific                                                   Activity Volume Activity                                                   Antioxidant Isotope Ligand mCi uL mCi/mg T = 0 T = 4 T = 24                 __________________________________________________________________________    GA 5 mg/mL                                                                           Y-90                                                                              N--Me                                                                              2.5 50  275 98  97 88                                           GA 25 mg/mL Y-90 N--Me 2.5 50 275 98 98 87                                    AA 5 Y-90 N--Me 3.7 50 850 100 100 92                                         AA 50 Y-90 N--Me 3.7 50 850 100 100 95                                      __________________________________________________________________________

The radioprotectants, e.g., gentisic acid or ascorbic acid, were addedeither upon completion of the radiolabeling procedure or as part of theradiolabeling reaction mixture. The preferred option was to employ theradioprotectant as part of the radiolabeling reaction mixture.

B. Y-90 Chelation Procedure. Carrier free ⁹⁰ YCl₃ (20-200 μL in 0.5NHCl) was obtained from DuPont (Wilmington, Del.). The carrier freemolecule was diluted with ammonium acetate buffer (0.5M, pH 5) to atotal volume of 0.4 mL. Fifty microliters (500 mg/mL) of ascorbic acidand 50-100 μL (10 mg/mL) of DOTA-biotin conjugate prepared in accordancewith Example XV(F) above are added to the buffered ⁹⁰ YCl₃ solution.Ascorbic acid is present as an antioxidant to prevent radiolysis of thepreparation as discussed above. The mixture was incubated for one hourat 80° C. Upon completion of the incubation, 55 μL of 100 mM DTPA(diethylenetriamine-pentaacetic acid) is added to the mixture to chelateany unbound Y-90. The final preparation was diluted to 10 mL with 0.9%NaCl.

C. Radiolysis product and oxidation. N-methyl-glycine-DOTA biotin wasexamined via HPLC for radiolysis in accordance with the followingprocedure. To a solution of 80 μg (0.1 pmol) of Y-90-labeledN-methyl-glycine-DOTA-biotin conjugate in 0.4 ml of 0.5M ammoniumacetate, pH 5, was added 43 μl of 23 mM (1.0 μmol) aqueous sodiumperiodate. The mixture was maintained at room temperature for 30 minutesprior to evaluation by HPLC as set forth above.

The oxidized biotin-containing conjugate exhibited the same HPLCretention time as the radiolysis product of the correspondingbiotin-containing conjugate. The oxidized biotin-containing conjugateretained the ability to bind avidin. This result was further confirmedby Y-90-radiolabeled N-methyl-glycine-DOTA-sulfoxide biotin, which wasprepared by reaction of N-methyl-glycine-DOTA-biotin with sodiumperiodate as set forth below. Consequently, the "radiolysis product" ofthe biotin-containing conjugate may represent an oxidizedbiotin-containing conjugate rather than a conjugate wherein Y-90 isescaping its DOTA chelate. Accordingly, the oxidized biotin-containingconjugate may be employed in the practice of the present invention.Radiolysis may result in total decomposition of the product conjugate,rather than merely oxidation as set forth above, however. In suchcircumstances, the use of radioprotectants will be beneficial.

Sulfoxobiotinyl-N-methyl-glycyl-aminobenzyl-DOTA. Sodium periodate (26mg) was added to a solution of biotinyl-N-methylglycyl-aminobenzyl-DOTA(10 mg) in water (1.0 ml). After the reaction was maintained at 23° C.for 30 minutes, NMR showed no remaining starting material as evidencedby downfield shift of imide methine from 4.4 and 4.6 ppm (startingmaterial) to 4.7 and 4.9 ppm (product) as well as by downfield shift ofS-methylene from 2.4 and 2.6 ppm (starting material) to 2.5 and 2.7 ppm(product). The product was purified by preparative HPLC. Analysis of thepurified product on analytical HPLC showed a single peak (7.69 minutes)having a 2 minute shorter retention time than the non-oxidized startingmaterial. Gradient 95% H₂ O.1TFA, 5% CH₃ CN to 50% H₂ O.1TFA, 50% CH₃ CNover 15 minutes.

Kits containing one or more of the components described above are alsocontemplated. For instance, radiohalogenated biotin may be provided in asterile container for use in pretargeting procedures. A chelate-biotinconjugate provided in a sterile container is suitable forradiometallation by the consumer; such kits would be particularlyamenable for use in pretargeting protocols. Alternatively,radiohalogenated biotin and a chelate-biotin conjugate may be vialed ina non-sterile condition for use as a research reagent.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. An article of manufacture comprising a packagehaving a label and containing a conjugate suitable for chelation with a+3 metal active agent, wherein the conjugate comprises a DOTA chelatecomponent, a biotin component and a linker component resistant tobiotinidase cleavage;and wherein, upon administration to a mammalianrecipient, the conjugate is capable of localizing to a previously targetsite-localized targeting moiety-streptavidin conjugate; and wherein thelabel identifies the DOTA, biotin, and linker conjugate components. 2.An article of manufacture of claim 1 wherein the label further indicatesthat the conjugate is limited to investigational use or indicates anindication for which the conjugate has been approved for use in humans.3. An article of manufacture of claim 1 wherein the conjugate iscontained within a vial.
 4. An article of manufacture of claim 3 whereinthe first conjugate is vialed in a sterile, pyrogen-free environment. 5.An article of manufacture of claim 2 wherein the indication is smallcell lung cancer and the targeting moiety binds to an antigen associatedwith small cell lung cancer.
 6. An article of manufacture of claim 5wherein the antigen is the NR-LU-10 antigen.
 7. An article ofmanufacture of claim 1 wherein the label further identifies an activeagent suitable for use with the conjugate, such active agent beingselected from the group consisting of Y-90, In-111, Tc-99m, Re-186,Re-188, Cu-67 and Lu-177.
 8. An article of manufacture of claim 1wherein the conjugate comprises a biotin-DOTA compound of the followingformula: ##STR50## wherein a linker L is selected from the groupcomprising: 1) a D-amino acid-containing linker of the formula ##STR51##2) a linker of the formula ##STR52## 3) a linker of the formula##STR53## and 4) a linker of the formula ##STR54## wherein L' isselected from the group comprising: a) --NH--CO--(CH₂)_(n) --O--;b)--NH--; c) ##STR55## d) --NH--CS--NH--; and e) --NH--CO--(CH₂)_(n)--NH--,wherein R¹ is hydrogen; lower alkyl; lower alkyl substituted withone or more hydrophilic groups including (CH₂)_(m) --OH, (CH₂)_(m)--OSO₃, (CH₂)_(m) --SO₃, and ##STR56## where m is 1 or 2;glucuronide-substituted amino acids; or other glucuronide derivatives;R² is hydrogen; lower alkyl; substituted lower alkyl having one or moresubstituents selected from the group comprising hydroxy, sulfate, andphosphonate; or a hydrophilic moiety; R³ is hydrogen; an amine; a loweralkyl; a hydroxy-, sulfate- or phosphonate-substituted lower alkyl; aglucuronide; or a glucuronide-derivatized amino acid; R⁴ is hydrogen,lower alkyl or ##STR57## R' is hydrogen; --(CH₂)₂ --OH or a sulfate orphosphonate derivative thereof; or ##STR58## R" is a bond or --(CH₂)_(n)--CO--NH--; and n ranges from 0-5.
 9. An article of manufacture of claim8 wherein L is a D-amino acid-incorporating linker of the formula##STR59##10.
 10. An article of manufacture of claim 9 wherein R¹ is CH₃and R² is H.
 11. An article of manufacture of claim 8 wherein L is alinker of the formula
 12. An article of manufacture of claim 11 whereinR³ is hydrogen; R⁴ is CH₃ ; and n is 4 or wherein R³ is hydrogen; R⁴ isCH₃ ; and n is
 0. 13. An article of manufacture of claim 10 wherein R³is hydrogen; R⁴ is and n is
 4. 14. An article of manufacture of claim 7wherein the active agent is Y-90 and the biotin component is oxidizedbiotin.